RU2807742C1 - Rsv f mutant protein and its application - Google Patents

Abstract

FIELD: biotechnology; medicine.

SUBSTANCE: mutant F protein of respiratory syncytial virus (RSV) having a mutation, where the mutation is a substitution of leucine corresponding to leucine at position 141 of the amino acid sequence SEQ ID NO: 1, or the leucine corresponding to the leucine at position 142 with a cysteine, and the replacement of the leucine corresponding to the leucine at position 373 with a cysteine, followed by the formation of a disulfide bond between the cysteines. The invention also relates to a fusion protein, a mutant protein multimer, an immunogenic product of particulate or immunogen, a pharmaceutical composition and a vaccine containing this mutant protein, the respiratory syncytial virus F protein.

EFFECT: effective treatment of RS viral infection.

39 cl, 35 dwg, 9 tbl, 9 ex

Description

Technical field

[0001]

The present invention relates to the mutant RSV F protein and its use.

Prior Art

[0002]

Respiratory syncytial virus (RSV) infection is a respiratory infection common throughout the world. Children under 5 years of age are at high risk of exacerbation of the disease. RSV infection causes lower respiratory tract inflammation in more than 30 million people each year, and more than 3 million people require hospitalization each year. Therefore, many efforts have now been made to develop a vaccine to prevent RSV infection. In the past, in 1965-66, clinical trials (subjects were infants) were conducted using formalin-inactivated virus as the vaccine antigen. However, instead of reducing the incidence of infectious diseases, this led to a 16-fold increase in the number of hospitalizations for RSV infection. One reason is that during inactivation treatment, F antigen, which is one of the surface antigens of the virus, is denatured and only antibodies with low RSV neutralizing activity are induced, and thus the immune response is suppressed. Although many attempts have been made since then to develop a vaccine, however, an effective vaccine has not yet been obtained.

[0003]

Accordingly, a study was conducted using sera from healthy adults and it was noted that severe RSV infection was rarely observed in healthy adults. As a result of the study, it was found that sera from healthy adults have a high ability to neutralize viral infection, and that the effect of neutralizing infection is due to an antibody against the RSV F protein (Non-Patent Document 1). Studies have also been conducted on the RSV F protein and it has been found that the mature F protein has a metastable conformation before fusion (which may be referred to herein as “pre-F” or “type F pre-protein”) and a most stable conformation after fusion (which may be referred to herein as “post-F” or “post-F type protein”), and, due to its instability, the prefusion conformation tends to refold early into the postfusion conformation in the bulk solution of the vaccine composition.

In addition, since the structure of pre-F, which was difficult to obtain stably, became clearer (Non-Patent Document 2), it was discovered that the F type pre-protein forms a trimer, and that some of the epitopes (epitopes Φ, I, II , III, IV, V, etc.) present in the F-type pre-protein are lost due to a structural change in the post-F.

[0004]

In a study that examined adult serum in more detail, it became clear that among the anti-F protein antibodies contained in serum, the antibodies with a high ability to neutralize viral infection are mainly antibodies that specifically bind to the Φ epitope and to the V epitope present in the type F pre-protein and that these antibodies specifically recognize the type F pre-protein but do not bind to post-F, and that the above serum also contains an antibody that also binds to post-F but has low neutralizing capacity viral infection (Non-Patent Document 3). In addition, an infant with insufficient immune system maturation is at high risk of worsening symptoms during infection. However, this is because an infant who has been infected for the first time or has a low frequency of infection has little ability to induce immunity targeting the Φ epitope or the V epitope. Effective induction of antibodies that have a high ability to neutralize viral infection in an antigen vaccine, is important, and it is believed that the ideal vaccine antigen is one that can preferentially induce antibodies against the Φ epitope or the V epitope with neutralizing ability.

[0005]

Attempts have been made to stabilize an F type pre-protein having a Φ epitope or a V epitope by mutation or the like that introduces an artificial disulfide bond into the RSV F protein. For example, there are reports of the DS-Cav1 mutant (Patent Document 1, Non-Patent Document 5, Non-Patent Document 6) or other mutants (Patent Document 2). However, stabilization of the F type pre-protein has been insufficient and research in this area is still ongoing.

Related documents

[Patent Document]

[0006]

[Patent Document 1] International Publication No. 2017/172890.

[Patent Document 2] International Publication No. 2017/109629.

[Non-patent documents]

[0007]

[Non-Patent Document 1] Ngwuta JO et al., Science Translational Medicine 2015; 309: 309ra162.

[Non-Patent Document 2] Jason S. McLellan et al., Science 2013; 340:1113.

[Non-Patent Document 3] Morgan SA Gilman et al., 2016, Science Immunology, 1(6), pii: eaaj1879.

[Non-Patent Document 4] Eileen Goodwin et al., Immunity 2018; 48(2):339.

[Non-Patent Document 5] Jason S. McLellan et al., Science 2013; 342:592.

[Non-Patent Document 6] M. Gordon Joyce et al., Nature structural and molecular biology 2016; 23 (9): 811.

[Description of the invention]

[Problems that can be solved by the present invention]

[0008]

The present invention relates to the development of a method for effectively inducing an antibody that has a high ability to neutralize RSV infection.

Solutions to these problems

[0009]

The present inventors have discovered that a mutant RSV F protein including a specific amino acid mutation effectively induces an antibody having a high ability to neutralize RSV infection, and on this basis the present invention has been developed.

The present invention includes:

[0010]

[1] An RSV F mutant protein having a mutation, wherein the mutation is a replacement of a leucine corresponding to leucine at position 141 of amino acid sequence SEQ ID NO: 1, or a leucine corresponding to leucine at position 142, with a cysteine, and a replacement of a leucine corresponding to leucine at position 373, to cysteine, followed by the formation of a disulfide bond between cysteines.

[2] Mutant RSV F protein according to [1], where the mutant RSV F protein is derived from RSV subtype A or RSV subtype B.

[3] Mutant F protein of RSV according to [2], where RSV subtype A is RSV A2 strain or long chain RSV strain.

[4] Mutant RSV F protein according to [2], where RSV subtype B is strain RSV 18537.

[5] A mutant RSV F protein according to any one of [1] to [4], having the ability to induce an antibody against the RSV F type pre-protein, and comprising an amino acid sequence wherein in an amino acid sequence that is 85% or more identical to SEQ ID NO : 2, the leucine corresponding to leucine at position 141, or the leucine corresponding to leucine at position 142, and the leucine corresponding to leucine at position 373 of the amino acid sequence SEQ ID NO: 1, are replaced by cysteines to form a disulfide bond between the cysteines.

[6] A mutant RSV F protein according to any one of [1]-[4], wherein, in addition, the amino acid forming a furin recognition site that is present on the C-terminal side of the pep27 region is replaced such that the furin recognition site is not recognized furin.

[7] The RSV F mutant protein according to [6], wherein the amino acid forming the furin recognition site is an amino acid selected from the group consisting of arginine corresponding to arginine at position 133, arginine corresponding to arginine at position 135, and arginine corresponding to arginine at position 136 of amino acid sequence SEQ ID NO: 1, and such amino acid is replaced by a non-basic amino acid.

[8] A mutant RSV F protein according to [6] or [7], having the ability to induce an antibody against the RSV F type pre-protein, and comprising an amino acid sequence wherein in an amino acid sequence that is 85% or more identical to SEQ ID NO: 3 , a leucine corresponding to leucine at position 141, or a leucine corresponding to leucine at position 142, and a leucine corresponding to leucine at position 373 of the amino acid sequence SEQ ID NO: 1, is replaced by cysteines and, in addition, an amino acid selected from the group consisting of arginine , corresponding to arginine at position 133, arginine corresponding to arginine at position 135, and arginine corresponding to arginine at position 136 of the amino acid sequence SEQ ID NO: 1, is replaced by a non-basic amino acid where a disulfide bond is formed between the cysteines.

[9] Mutant RSV F protein according to [7] or [8], wherein the non-essential amino acid is asparagine.

[10] The RSV F mutant protein according to any one of [1] to [9], wherein, in addition, the threonine corresponding to the threonine at position 189 of amino acid sequence SEQ ID NO: 1 and/or the serine corresponding to the serine at position 190 are replaced hydrophobic amino acids.

[11] Mutant RSV F protein according to [10], wherein each hydrophobic amino acid is independently selected from the group consisting of valine, isoleucine and leucine.

[12] The RSV F mutant protein according to any one of [1]-[11], wherein, in addition, the lysine corresponding to lysine at position 42 of amino acid sequence SEQ ID NO: 1 is replaced by arginine, and/or valine corresponding to valine at position 384, replaced by threonine.

[13] An RSV F mutant protein having a mutation, wherein the mutation is a replacement of glutamic acid corresponding to glutamic acid at position 60 of the amino acid sequence SEQ ID NO: 1 with a non-acidic amino acid.

[14] The RSV F mutant protein according to [13], wherein the non-acidic amino acid is selected from the group consisting of methionine, phenylalanine, leucine, threonine and serine.

[15] A fusion protein comprising: a mutant RSV F protein according to any one of [1]-[14]; and a multimerization domain fused to the C terminus of the mutant RSV F protein.

[16] A fusion protein according to [15], where the multimerization domain is a foldon domain.

[17] A fusion protein according to [16], containing the amino acid sequence of any of SEQ ID NO: 10-13.

[18] A multimer of a mutant RSV F protein according to any one of [1]-[14], wherein the fusion protein according to any one of [15]-[17] is linked through a multimerization domain.

[19] Multimer according to [18], which is a trimer.

[20] A particle product of a mutant RSV F protein according to any of [1] - [14] or a multimer according to any of [15] - [17], wherein the mutant RSV F protein or fusion protein contains a particle-forming domain, two or more mutant RSV F protein or multimer aggregate through the particle-forming domain to form a particle, and this particle-forming domain is located at a position closer to the I epitope than to the Φ epitope on the steric structure of the mutant RSV F protein or multimer.

[21] A particulate product according to [20], wherein two or more mutant RSV F proteins or fusion proteins to produce a particulate product, wherein the particulate domain is linked to the C-terminus of the mutant RSV F protein or fusion protein, aggregate through a particle-forming domain.

[22] A particulate product according to [20] or [21], wherein the multimer is a trimer consisting of a fusion protein according to [16].

[23] The particulate product according to [22], wherein the particulate domain is an Fc domain consisting of the amino acid sequence of SEQ ID NO: 30, and the particulate domain is aggregated by binding to the Z domain of protein A immobilized on the VLP, derived from a modified HBs antigen.

[24] The particulate product according to [22], wherein the particulate domain is a peptide consisting of the amino acid sequence SEQ ID NO: 42.

[25] A fusion protein for producing a particulate product, comprising: a mutant RSV F protein according to any one of [1]-[14] or a fusion protein according to any one of [15]-[17], and a particulate domain fused to it C-end.

[26] A fusion protein for producing a particulate product according to [25], wherein the particulate domain is an Fc domain consisting of the amino acid sequence SEQ ID NO: 30.

[27] A fusion protein for producing a particulate product according to [25], wherein the particulate domain is a peptide consisting of the amino acid sequence SEQ ID NO: 42.

[28] A polynucleotide encoding a mutant RSV F protein according to any of [1]-[14], a fusion protein according to any of [15]-[17], or a fusion protein to produce a particulate product according to any of [25]-[ 27].

[29] An expression cluster containing a polynucleotide according to [28].

[30] A host cell containing an expression cluster according to [29].

[31] An immunogen comprising: a mutant RSV F protein according to any one of [1]-[14], a fusion protein according to any one of [15]-[17], a multimer according to any one of [18]-[19], or a product in the form particles according to any of [20]-[24].

[32] Pharmaceutical composition containing an expression cluster according to [29] or an immunogen according to [31].

[33] RSV vaccine containing an expression cluster according to [29] or an immunogen according to [31].

[34] Pharmaceutical composition according to [32] or RSV vaccine according to [33], intended for administration to humans.

Invention effect

[0011]

In accordance with the present invention, a method for effectively inducing an antibody having a high ability to neutralize RSV infection can be developed. The mutant F protein of RSV, which has significantly higher stability compared to the normal protein, can serve as a type F pre-protein. The mutant RSV F protein of the invention has the effect of inducing a group of antibodies that are considered to have a high ability to neutralize the virus, compared to a group of antibodies that are considered to be less effective in neutralizing the virus, especially in humans (hereinafter, this effect is referred to as the effect of preferentially inducing a group of antibodies that are thought to have a high ability to neutralize the virus).

Brief description of drawings

[0012]

[Fig. 1] Graph illustrating the storage stability of the Φ epitope in Example 1 according to one embodiment of the present invention.

[Fig. 2] Graph illustrating the storage stability of the Φ epitope in Example 2 according to one embodiment of the present invention.

[Fig. 3] Graph illustrating trimerization in Example 2 according to one embodiment of the present invention.

[Fig. 4] Graph illustrating the storage stability of the Φ epitope in Example 3 according to one embodiment of the present invention.

[Fig. 5] Graph illustrating trimerization in Example 3 according to one embodiment of the present invention.

[Fig. 6] Graph illustrating the expression level in Example 3 according to one embodiment of the present invention.

[Fig. 7] Graph illustrating the storage stability of the Φ epitope in Example 4 according to one embodiment of the present invention.

[Fig. 8] Graph illustrating trimerization in Example 4 according to one embodiment of the present invention.

[Fig. 9] Graph illustrating the storage stability of the Φ epitope in Example 5 according to one embodiment of the present invention.

[Fig. 10] Graph illustrating trimerization in Example 5 according to one embodiment of the present invention.

[Fig. 11] Graph illustrating the Φ epitope recognition property of Example 5 in accordance with one embodiment of the present invention.

[Fig. 12] Graph illustrating the results of virus neutralizing antibody adsorption tests in human sera in Example 5 according to one embodiment of the present invention.

[Fig. 13] Graph illustrating the results of virus neutralizing antibody adsorption tests in human sera in Example 5 according to one embodiment of the present invention.

[Fig. 14] Graph illustrating the results of tests for antibodies induced by murine immunity in Example 5, according to one embodiment of the present invention.

[Fig. 15] Graph illustrating the storage stability of the Φ epitope in Example 6 according to one embodiment of the present invention.

[Fig. 16] Graph illustrating trimerization in Example 6 according to one embodiment of the present invention.

[Fig. 17] Graph illustrating the recognition property of epitope I in Example 6 according to one embodiment of the present invention.

[Fig. 18] Graph illustrating the results of tests for antibodies induced by murine immunity in Example 6, according to one embodiment of the present invention.

[Fig. 19] Graph illustrating the storage stability of the Φ epitope in Example 7 according to one embodiment of the present invention.

[Fig. 20] Graph illustrating trimerization in Example 7 according to one embodiment of the present invention.

[Fig. 21] Graph illustrating the recognition property of epitope I in Example 7 according to one embodiment of the present invention.

[Fig. 22] Graph illustrating the storage stability of the Φ epitope in Example 7 according to one embodiment of the present invention (linker insertion assay).

[Fig. 23] Graph illustrating trimerization in Example 7 according to one embodiment of the present invention (linker insertion assay).

[Fig. 24] Graph illustrating the recognition property of epitope I in Example 7 according to one embodiment of the present invention (linker insertion assay).

[Fig. 25] Graph showing particle diameters in Example 7 according to one embodiment of the present invention.

[Fig. 26] Graph illustrating the results of polyacrylamide gel electrophoresis in Example 8 according to one embodiment of the present invention (Part 1).

[Fig. 27] Graph illustrating the results of polyacrylamide gel electrophoresis in Example 8 according to one embodiment of the present invention (Part 2).

[Fig. 28] Graph illustrating the storage stability of the Φ epitope in Example 8 according to one embodiment of the present invention.

[Fig. 29] Graph illustrating trimerization in Example 8 according to one embodiment of the present invention.

[Fig. 30] Graph illustrating the recognition property of epitope I in Example 8 according to one embodiment of the present invention.

[Fig. 31] Graph showing particle diameters in Example 8 according to one embodiment of the present invention.

[Fig. 32] Graph illustrating the results of tests for antibodies induced by murine immunity in Example 8, according to one embodiment of the present invention.

[Fig. 33] Graph illustrating the results of polyacrylamide gel electrophoresis in Example 9 according to one embodiment of the present invention.

[Fig. 34] Graph illustrating the storage stability of the Φ epitope in Example 9 according to one embodiment of the present invention.

[Fig. 35] Graph illustrating the recognition property of epitope I in Example 9 according to one embodiment of the present invention.

Method for carrying out the invention

[0013]

One embodiment of the present invention is a mutant RSV F protein having a mutation, wherein the mutation is a substitution of a leucine corresponding to leucine at position 141 of amino acid sequence SEQ ID NO: 1, or a leucine corresponding to leucine at position 142, with a cysteine, and a substitution of a leucine corresponding to leucine at position 373, to cysteine, and where a disulfide bond is formed between the cysteines.

[0014]

The natural RSV F protein is first expressed as an F0 (precursor) polypeptide. The F0 polypeptide is processed after cleavage of the endoplasmic reticulum migration signal by the intracellular furin-like protease at two sites to form the F1 polypeptide, the F2 polypeptide and the Pep27 polypeptide. In this case, the Pep27 polypeptide is excised and thus does not form part of the mature F protein. The F2 polypeptide is derived from the N-terminal side of the F0 polypeptide and is linked to the F1 polypeptide by two disulfide bonds. The F1 polypeptide is derived from the C-terminal side of the F0 polypeptide and immobilizes the mature F protein to the membrane via a transmembrane domain. The three protomers of the F2-F1 heterodimer form the mature F protein as a trimer, which is an F type pre-protein that functions to fuse the viral membrane and the target cell membrane.

[0015]

The RSV F mutant protein of the invention comprises an F2 polypeptide and an F1 polypeptide at the N-terminal side. As the F1 polypeptide, it is preferable to use an F1 polypeptide that does not include a transmembrane region and an intracellular region (hereinafter may be referred to as an F1 polypeptide (extracellular region)). In addition, the mutant RSV F protein of the invention may include a Pep27 polypeptide on the N-terminal side of the F1 polypeptide by removing one of the furin-like protease recognition sites through a mutation that will be described below. In addition, a signal sequence at the N-terminal side of the F2 polypeptide, a linker between the F1 polypeptide and the F2 polypeptide, and a tag used for purification, or the like may also be included. on the C-terminal side. In addition, the RSV mutant F protein of the invention preferably forms a trimer.

[0016]

The RSV F mutant protein of the invention is preferably derived from RSV subtype A or RSV subtype B.

Specific examples of RSV subtype A include RSV strain A2, long-chain RSV strain, RSV S2 strain, and RSV lineage 19 strain, and among these, RSV A2 strain or long-chain RSV strain is preferred.

Specific examples of RSV subtype B include RSV strain 18537, RSV strain B1, and RSV strain 9320, and among them, RSV strain 18537 is preferred.

[0017]

The amino acid sequence of SEQ ID NO: 1 is the amino acid sequence of the F0 polypeptide of the RSV A2 strain F protein. In this regard, the structural elements of the F0 polypeptide are described.

In SEQ ID NO: 1, the amino acid sequence of the signal sequence is the amino acid sequence at positions 1-25.

In SEQ ID NO: 1, the amino acid sequence of the F2 polypeptide is the amino acid sequence at positions 26-109.

In SEQ ID NO: 1, the amino acid sequence of the pep27 region is the amino acid sequence at positions 110-136.

In SEQ ID NO: 1, the amino acid sequence of the F1 polypeptide (extracellular region) is the amino acid sequence at positions 137-513.

In SEQ ID NO: 1, the amino acid sequence of the F1 polypeptide (transmembrane region and intracellular region) is the amino acid sequence at positions 514-574. The amino acid sequence of the F1 polypeptide (transmembrane region and intracellular region) may be a sequence that is 85% or more, 90% or 95% or more identical to the amino acid sequence at positions 514-574 of SEQ ID NO: 1.

[0018]

The RSV F protein may include an F2 polypeptide and an F1 polypeptide (extracellular region). An example of an RSV F protein that includes an F2 polypeptide and an F1 polypeptide (extracellular region) is the amino acid sequence of SEQ ID NO: 2, which includes, in that order, the amino acid sequence at positions 26-109 of SEQ ID NO: 1 and the amino acid sequence at positions 137-513.

The RSV F protein may include an F2 polypeptide and an F1 polypeptide (extracellular region) to which the pep27 region is associated. An example of an RSV F protein that includes, in that order, an F2 polypeptide and an F1 polypeptide (the extracellular region) to which the pep27 region is associated is the amino acid sequence of SEQ ID NO: 3, which includes the amino acid sequence at positions 26-513 of SEQ ID NO: 1.

These polypeptides may be individual polypeptide chains and are preferably linked by disulfide bonds. That is, the RSV F protein may be a complex of such polypeptides.

[0019]

Mutation in which leucine is replaced by cysteine

The RSV F mutant protein of the invention comprises a mutation, namely, the substitution of a leucine corresponding to leucine at position 141 of amino acid sequence SEQ ID NO: 1, or a leucine corresponding to leucine at position 142, with a cysteine, and a substitution of a leucine corresponding to leucine at position 373, to cysteine. In the mutant RSV F protein having the above amino acid mutation, in addition to the naturally occurring disulfide bond between the introduced cysteines, a new disulfide bond is formed, and thus the effect of increasing the storage stability (preservation of the pre-type) of the F epitope is achieved. To form a trimer in particular, the leucine corresponding to leucine at position 141 of SEQ ID NO: 1 is replaced by cysteine, and the leucine corresponding to leucine at position 373 is replaced by cysteine.

[0020]

In the present specification, the mutation position of the mutant RSV F protein is indicated by the amino acid number of the reference sequence represented by SEQ ID NO: 1.

[0021]

Leucine corresponding to leucine at position 141 of amino acid sequence SEQ ID NO: 1, leucine corresponding to leucine at position 142, and leucine corresponding to leucine at position 373 refer to leucine corresponding to leucine at position 141, leucine corresponding to leucine at position 142, and leucine corresponding to leucine at position 373 in the amino acid sequence of SEQ ID NO: 1, regardless of the origin of the mutant RSV F protein. That is, for the amino acid sequence of the RSV F protein to which a mutation is to be introduced, if an alignment is made with the amino acid sequence of SEQ ID NO: 1, these amino acids refer to the leucine residues respectively located at the leucine position 141, at the leucine 142 position and at the leucine 373 amino acid sequence SEQ ID NO: 1.

Thus, for example, in the case of the amino acid sequence of the mutant RSV F protein, the N-terminal side of which is one amino acid shorter compared to the amino acid sequence of SEQ ID NO: 1, the leucine at position 141 in the amino acid sequence of SEQ ID NO: 1 is at position 140, and such leucine may be referred to as leucine corresponding to leucine at position 141 in the amino acid sequence of SEQ ID NO: 1.

For the amino acid sequence of the RSV F protein to which a mutation is to be introduced, when aligned with the amino acid sequence of SEQ ID NO: 1, in the case where an amino acid other than leucine is present at the leucine position corresponding to leucine at position 141, at the leucine position, corresponding to leucine at position 142, and/or leucine at position 373 of the amino acid sequence SEQ ID NO: 1, then the RSV F mutant protein of the invention also includes a variant in which the amino acid is replaced by cysteine. Moreover, in the case where the non-leucine amino acid is cysteine, the cysteine substitution is not required and the amino acid may itself be cysteine.

[0022]

One embodiment of the invention is a mutant RSV F protein that includes an F2 polypeptide and an F1 polypeptide (extracellular region) derived from a mutant F0 polypeptide that includes an amino acid sequence wherein there is a leucine at position 141 or a leucine at position 142, and a leucine at position 373 are replaced by cysteines in the amino acid sequence of SEQ ID NO: 1, and where a disulfide bond is formed between the cysteines. Specifically, this embodiment includes an amino acid sequence wherein in the amino acid sequence of SEQ ID NO: 2, leucine at position 89 or leucine at position 90 and leucine at position 321 are replaced by cysteines.

[0023]

One embodiment of the invention is a mutant RSV F protein having the ability to induce an antibody against the RSV F type pre-protein, and comprising an F2 polypeptide and an F1 extracellular region polypeptide that are produced from a mutant F0 polypeptide having an amino acid sequence that is 85% or more , preferably 90% or more, and more preferably 95% or more identical to the amino acid sequence of SEQ ID NO: 1, wherein a leucine corresponding to leucine at position 141, or a leucine corresponding to leucine at position 142, and a leucine corresponding to leucine at position 373 amino acid sequence SEQ ID NO: 1, replaced by cysteines, and where a disulfide bond is formed between the cysteines. Specifically, this embodiment includes an amino acid sequence that is 85% or more, preferably 90% or more, and more preferably 95% or more identical to the amino acid sequence of SEQ ID NO: 2, wherein leucine corresponding to leucine at position 141 (at position 89 in SEQ ID NO: 2), or leucine corresponding to leucine at position 142 (at position 90 in SEQ ID NO: 2), and leucine corresponding to leucine at position 373 (at position 321 in SEQ ID NO: 2) amino acid sequences SEQ ID NO: 1, replaced by cysteines.

[0024]

As used herein, "percentage (%) identity" with respect to an amino acid sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical to the amino acid residues of a particular reference polypeptide sequence after aligning those sequences, introducing spaces if necessary to obtain maximum % identity, however, any conservative substitution is not considered when determining sequence identity. Alignment to estimate % identity can be achieved using various methods known to those skilled in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. One skilled in the art can determine appropriate parameters for sequence alignment, including any algorithm necessary to achieve maximum alignment over the entire length of the sequences being compared. However, in this case, the % identity value is obtained using the BLAST sequence comparison computer program in pairwise alignment. If BLAST is used to compare amino acid sequences, then the % identity of a given amino acid sequence A with a given amino acid sequence B is calculated using the formula:

100 • X/Y

where X is the number of amino acid residues with identity scores as determined by aligning A and B according to the BLAST sequence alignment program, and Y is the total number of amino acid residues of B. If the length of the amino acid sequence of A is different from the length of the amino acid sequence sequence B, it is understood that the % identity of A with respect to B is different from the % identity of B with respect to A. Unless otherwise stated, in this application, all % identity values are obtained using the computer program BLAST, as described in the previous paragraph.

[0025]

An amino acid sequence having a predetermined identity with a predetermined amino acid sequence is the result of a substitution, deletion, insertion, or addition of amino acids to a predetermined amino acid sequence. The replacement is preferably a conservative replacement. A "conservative substitution" is where an amino acid residue is replaced with another chemically similar amino acid residue so that the activity of the protein is not substantially changed. Examples of such substitutions include the case where a hydrophobic residue is replaced by another hydrophobic residue; and the case where a polar residue is replaced by another polar residue having a charge, etc. Examples of functionally similar amino acids suitable for such substitutions include non-polar (hydrophobic) amino acids such as alanine, valine, isoleucine, leucine, proline, tryptophan, phenylalanine, methionine, and the like. Examples of polar (neutral) amino acids are glycine, serine, threonine, tyrosine, glutamine, asparagine, cysteine, etc. Examples of positively charged (basic) amino acids are arginine, histidine, lysine, etc. Moreover, examples of negatively charged (acidic) amino acids are aspartic acid, glutamic acid and the like.

[0026]

Glutamic acid mutation at position 60

Another variant of the RSV F mutant protein of the invention is an RSV F mutant protein having a mutation, wherein the mutation is a replacement of glutamic acid corresponding to glutamic acid at position 60 of amino acid sequence SEQ ID NO: 1 with a non-acidic amino acid.

[0027]

The non-acidic amino acid is not particularly limited provided that the mutant RSV F protein will achieve the effect of preferentially inducing a group of antibodies that are believed to contribute significantly to improving the storage stability of the Φ epitope, promoting trimerization and neutralizing the virus. However, specific examples of such amino acids include neutral amino acids, basic amino acids, and non-polar (hydrophobic) amino acids. More specifically, examples of neutral amino acids are glycine, serine, threonine, tyrosine, glutamine, asparagine and cysteine; examples of basic amino acids are arginine, histidine and lysine; and examples of non-polar (hydrophobic) amino acids are alanine, valine, isoleucine, leucine, proline, tryptophan, phenylalanine and methionine.

The non-acidic amino acid is preferably a neutral amino acid or a non-polar (hydrophobic) amino acid.

Moreover, among neutral amino acids, threonine and serine are preferred, and among non-polar (hydrophobic) amino acids, amino acids selected from the group consisting of methionine, phenylalanine and leucine are preferred. Therefore, the non-acidic amino acid is preferably selected from the group consisting of methionine, phenylalanine, leucine, threonine and serine.

[0028]

One embodiment of the invention is a mutant RSV F protein that includes an F2 polypeptide and an F1 polypeptide (extracellular region) derived from a mutant F0 polypeptide that includes an amino acid sequence wherein glutamic acid at position 60 of amino acid sequence SEQ ID NO: 1 is replaced by a non- acidic amino acid. Specifically, included is an amino acid sequence wherein in the amino acid sequence of SEQ ID NO: 2, glutamic acid at position 35 is replaced by a non-acidic amino acid.

[0029]

One embodiment of the present invention is a mutant RSV F protein having the ability to induce an antibody against the RSV F type pre-protein, and comprising an F2 polypeptide and an F1 polypeptide (extracellular region), which are produced from a mutant F0 polypeptide having an amino acid sequence wherein the amino acid a sequence that is 85% or more, preferably 90% or more, and more preferably 95% or more identical to the amino acid sequence of SEQ ID NO: 1, glutamic acid, corresponding to glutamic acid at position 60 of the amino acid sequence of SEQ ID NO: 1, replaced by a non-acidic amino acid. Specifically included is an amino acid sequence, wherein in an amino acid sequence that is 85% or more, preferably 90% or more, and more preferably 95% or more identical to the amino acid sequence of SEQ ID NO: 2, glutamic acid corresponding to glutamic acid in position 60 (at position 35 in SEQ ID NO: 2) of the amino acid sequence SEQ ID NO: 1 is replaced with a non-acidic amino acid.

[0030]

A glutamic acid mutation at position 60 can be introduced along with the mutations described above where leucines are replaced by cysteines.

[0031]

Mutation of the furin recognition site

The mutant RSV F protein of the invention may be a mutant RSV F protein wherein, in addition, the amino acid forming a furin recognition site on the C-terminal side of the pep27 region is replaced so that the furin recognition site is not recognized by furin. With this amino acid substitution, the mutant RSV F protein has an F2 polypeptide and an F1 polypeptide (extracellular region) to which the pep27 region is associated, and thus achieves the effect of promoting trimerization while maintaining the stability of the F epitope during storage.

[0032]

The pep27 region is a region corresponding to positions 110 to 136 of amino acid sequence SEQ ID NO: 1, and the first recognition site on its N-terminal side and the second recognition site on its C-terminal side are cleaved by furin in the cell. The N-terminal side of the pep27 region is an F2 polypeptide and the C-terminal side is an F1 polypeptide, and cleavage produces an F2 polypeptide and an F1 polypeptide, and these polypeptides form a disulfide bond between cysteine residues, which is respectively inherent in the polypeptides when forming a complex. Therefore, typically, the RSV F protein does not include the pep27 region. However, due to the mutation, the second recognition site is not cleaved and only the first recognition site, present between the C-terminal side of the F2 polypeptide and the pep27 region, is cleaved by furin. As a result of this cleavage, a polypeptide is formed including the F1 polypeptide, to which the pep27 region is associated, and the F2 polypeptide, and these polypeptides form a disulfide bond between cysteine residues, which is accordingly characteristic of these polypeptides when forming a complex. Therefore, this embodiment according to the invention may be a complex that includes a polypeptide fragment containing an F1 polypeptide, to which the pep27 region is associated, and an F2 polypeptide.

[0033]

The substitution may be a change in one or more amino acids that form furin recognition sites. In particular, the amino acids that form the furin recognition sites are one or more amino acids selected from the group consisting of arginine corresponding to arginine at position 133, arginine corresponding to arginine at position 135, and arginine corresponding to arginine at position 136 of the amino acid sequence SEQ ID NO: 1, and these amino acids are preferably replaced with non-essential amino acids.

The arginine corresponding to arginine at position 133, the arginine corresponding to arginine at position 135, and the arginine corresponding to arginine at position 136 of the amino acid sequence SEQ ID NO: 1 can be independently replaced by a non-basic amino acid.

[0034]

The non-basic amino acid is not particularly limited as long as the effect of promoting trimerization is maintained while the Φ epitope remains stable during storage. However, particular examples of such amino acids are neutral amino acids, acidic amino acids and non-polar (hydrophobic) amino acids. More specifically, examples of neutral amino acids are glycine, serine, threonine, tyrosine, glutamine, asparagine and cysteine; examples of acidic amino acids are aspartic acid and glutamic acid; and examples of non-polar (hydrophobic) amino acids are alanine, valine, isoleucine, leucine, proline, tryptophan, phenylalanine and methionine. Among them, neutral amino acids are preferred, and asparagine is more preferred.

[0035]

One variant of the mutant RSV F protein of the invention comprises an amino acid sequence wherein, in the amino acid sequence of SEQ ID NO: 3, leucine at position 116 or leucine at position 117 and leucine at position 348 are replaced by cysteines, and an amino acid selected from the group consisting of arginine at position 108, arginine at position 110 and arginine at position 111, replaced by a non-essential amino acid.

[0036]

One variant of the mutant RSV F protein of the invention comprises an amino acid sequence wherein, in an amino acid sequence that is 85% or more, preferably 90% or more, and more preferably 95% or more identical to the amino acid sequence of SEQ ID NO: 3, leucine, corresponding to the leucine at position 141 (leucine at position 116 in SEQ ID NO: 3), or the leucine corresponding to the leucine at position 142 (leucine at position 117 in SEQ ID NO: 3), and the leucine corresponding to the leucine at position 373 (leucine at position 348 in SEQ ID NO: 3) the amino acid sequence of SEQ ID NO: 1, replaced by cysteines, and an amino acid selected from the group consisting of arginine corresponding to arginine at position 133 (arginine at position 108 in SEQ ID NO: 3), arginine , corresponding to arginine at position 135 (arginine at position 110 in SEQ ID NO: 3), and arginine corresponding to arginine at position 136 (arginine at position 111 in SEQ ID NO: 3) of the amino acid sequence SEQ ID NO: 1, replaced by non- basic amino acid.

[0037]

Another variant of the mutant RSV F protein of the invention comprises an amino acid sequence wherein, in the amino acid sequence SEQ ID NO: 3, glutamic acid at position 35 is replaced by a non-acidic amino acid, and an amino acid selected from the group consisting of arginine at position 108, arginine at position 110 and arginine at position 111, replaced by a non-basic amino acid.

[0038]

Another variant of the mutant RSV F protein of the invention comprises an amino acid sequence wherein, in an amino acid sequence that is 85% or more, preferably 90% or more, and more preferably 95% or more identical to the amino acid sequence of SEQ ID NO: 3, glutamic acid , corresponding to glutamic acid at position 60 in the amino acid sequence of SEQ ID NO: 1 (glutamic acid at position 35 in SEQ ID NO: 3) is replaced by a non-acidic amino acid, and an amino acid selected from the group consisting of arginine corresponding to arginine at position 133 (arginine at position 108 in SEQ ID NO: 3), arginine corresponding to arginine at position 135 (arginine at position 110 in SEQ ID NO: 3), and arginine corresponding to arginine at position 136 (arginine at position 111 in SEQ ID NO: 3) : 3) amino acid sequence SEQ ID NO: 1, replaced by a non-basic amino acid.

[0039]

In SEQ ID NO: 3, or in an amino acid sequence that is 85% or more, preferably 90% or more, and more preferably 95% or more identical to SEQ ID NO: 3, the pep27 region may be replaced by an artificial linker. The artificial linker sequence is not particularly limited, but its length is preferably 6-27 amino acid residues, with a sequence containing Gly and/or Ser being preferred. More specifically, examples thereof include Gly-Ser-Gly-Ser-Gly-Ser (SEQ ID NO: 18), (Gly-Gly-Gly-Gly-Ser) ₂ (SEQ ID NO: 19) and (Gly-Gly- Gly-Gly-Ser) ₃ (SEQ ID NO: 20).

[0040]

Other mutations

The mutant RSV F protein of the invention may be a mutant RSV F protein, wherein, in addition to a mutation in which a leucine corresponding to leucine at position 141, or a leucine corresponding to leucine at position 142, and a leucine corresponding to leucine at position 373 of the amino acid sequence SEQ ID NO: 1 is replaced by cysteines (hereinafter referred to as the mutation in which leucines are replaced by cysteines), there is a glutamic acid mutation at position 60 and/or a furin recognition site mutation described above, and in addition, an amino acid corresponding to threonine at position 189, and/or the amino acid corresponding to serine at position 190 of amino acid sequence SEQ ID NO: 1, are replaced by hydrophobic amino acids.

The mutant RSV F protein according to an embodiment of the invention has the effect that the level of expression in the expression system is significantly increased, while also maintaining the effect of excellent stability of the Φ epitope during storage and stimulation of trimerization, as well as the effect of preferentially inducing a group of antibodies that, are believed to provide a high degree of virus neutralization.

The amino acid corresponding to threonine at position 189 and the amino acid corresponding to serine at position 190 of the amino acid sequence SEQ ID NO: 1 can be independently replaced with a hydrophobic amino acid.

In addition, the RSV F mutant protein containing the substitution is difficult to recognize by an antibody that is ineffective or poor in neutralizing viral infection, and in addition has the effect of significantly increasing the level of expression in the expression system, maintaining excellent stability of the Φ epitope during storage and promoting trimerization, as well as the effect of preferentially inducing a group of antibodies that are believed to provide a high degree of neutralization of the virus. Conversely, if an antibody is induced that is ineffective or poor at neutralizing a viral infection, then there is an expected risk that an immune cell that contributes little or nothing to neutralize the viral infection will be activated, leading to an exacerbation of symptoms. However, as can be seen from the examples described below, the mutant RSV F protein according to an embodiment of the invention is difficult to recognize by an antibody that is ineffective or poorly neutralizes the viral infection, and thus the risk in the case of the mutant RSV F protein according to the invention will be lower. than with a conventional vaccine.

As a specific example, the mutant F protein of RSV according to an embodiment of the invention is difficult to recognize by an antibody that recognizes epitope I. Since epitope I induces an antibody that has a very low ability to neutralize viral infection, it is expected that there is a risk that epitope I will be recognized by the antibody , and the immune cell will hardly neutralize the viral infection, will increase, which will thus lead to an exacerbation of symptoms. However, as can be seen from the examples described below, in the case of using a mutant RSV F protein according to an embodiment of the invention, which is difficult to recognize by an antibody recognizing epitope I, the risk will be lower than in the case of a conventional vaccine.

[0041]

The hydrophobic amino acid is not particularly limited provided that in addition to the effect of significantly increasing the level of expression in the expression system, maintaining excellent stability of the Φ epitope during storage and promoting trimerization, as well as the effect of preferentially inducing a group of antibodies that are believed to provide a high degree of virus neutralization, this mutant RSV F protein will be difficult to recognize by an antibody that is ineffective or poor at neutralizing viral infection. However, specific examples of such amino acids include alanine, valine, isoleucine, leucine, proline, tryptophan, phenylalanine and methionine. Among them, amino acids selected from the group consisting of valine, isoleucine and leucine are preferred.

[0042]

Position 189 in SEQ ID NO: 1 corresponds to the amino acid at position 137 in SEQ ID NO: 2, and position 190 in SEQ ID NO: 1 corresponds to the amino acid at position 138 in SEQ ID NO: 2. In SEQ ID NO: 2, or in amino acid sequence that is 85% or more, 90% or more, or 95% or more identical to SEQ ID NO: 2, a mutation of one or both of these amino acids may be introduced in addition to a mutation in which leucines are replaced by cysteines and/or mutation of glutamic acid at position 60 described above.

In addition, position 189 in SEQ ID NO: 1 corresponds to the amino acid at position 164 in SEQ ID NO: 3, and position 190 in SEQ ID NO: 1 corresponds to the amino acid at position 165 in SEQ ID NO: 3. In SEQ ID NO: 3 , or in an amino acid sequence that is 85% or more, 90% or more, or 95% or more identical to SEQ ID NO: 3, a mutation of one or both of these amino acids may be introduced in addition to a mutation in which leucines are replaced by cysteines , mutations of glutamic acid at position 60, and/or mutations of the furin recognition site, as described above.

[0043]

One embodiment of the present invention may be a mutant RSV F protein in which, in addition to the mutation in which leucines are replaced by cysteines, there is a mutation of glutamic acid at position 60 and/or a mutation of the furin recognition site as described above, and in which, in amino acid sequence SEQ ID NO: 1, the amino acid corresponding to lysine at position 42 is replaced by arginine, and/or the amino acid corresponding to valine at position 384 is replaced by threonine.

The mutant RSV F protein according to an embodiment of the invention provides the effect that the expression level in the expression system is significantly increased, while also maintaining the effect of excellent storage stability of the Φ epitope and stimulation of trimerization, as well as the effect of preferentially inducing a group of antibodies that, are believed to have a high ability to neutralize the virus.

[0044]

Position 42 in SEQ ID NO: 1 corresponds to the amino acid at position 17 in SEQ ID NO: 2, and position 384 in SEQ ID NO: 1 corresponds to the amino acid at position 332 in SEQ ID NO: 2. In SEQ ID NO: 2 or in the amino acid sequence that is 90% or more identical to SEQ ID NO: 2, a mutation of one or both of these amino acids may be introduced in addition to the mutation in which leucines are replaced by cysteines and/or the glutamic acid mutation at position 60 described above, and in addition to the mutation at position 189 and/or position 190 as described above.

In addition, position 42 in SEQ ID NO: 1 corresponds to amino acid at position 17 in SEQ ID NO: 3, and position 384 in SEQ ID NO: 1 corresponds to amino acid at position 359 in SEQ ID NO: 3. In SEQ ID NO: 3 or in an amino acid sequence that is 90% or more identical to SEQ ID NO: 3, a mutation of one or both of these amino acids may be introduced in addition to a mutation in which leucines are replaced by cysteines, a glutamic acid mutation at position 60, and/or a site mutation recognition by furin as described above, and in addition to mutation at position 189 and/or position 190, as described above.

[0045]

The mutant RSV F protein may further include commonly known mutations, provided that these mutations do not interfere with the effects of the mutant RSV F protein. Thus, for example, a mutation may further be included to increase the level of expression in the expression system. Specifically, one or more substitutions selected from the group consisting of replacing the amino acid corresponding to proline at position 102 of amino acid sequence SEQ ID NO: 1 with alanine, replacing the amino acid corresponding to isoleucine at position 379 with valine, and replacing the amino acid with valine may be included. , corresponding to methionine at position 447, valine.

[0046]

Position 102 in SEQ ID NO: 1 corresponds to amino acid position 77 in SEQ ID NO: 2, position 379 in SEQ ID NO: 1 corresponds to amino acid position 327 in SEQ ID NO: 2, and position 447 in SEQ ID NO: 1 corresponds to amino acid at position 395 in SEQ ID NO: 2. In SEQ ID NO: 2 or in an amino acid sequence that is 85%, 90% or 95% or more identical to SEQ ID NO: 2, a mutation of one or both of these amino acids may be introduced in addition to the mutation in which leucines are replaced by cysteines, and/or the glutamic acid mutation at position 60 described above, and further, in addition to the mutation at position 189 and/or at position 190, or at position 42, and /or at position 384 as described above.

In addition, position 102 in SEQ ID NO: 1 corresponds to amino acid at position 77 in SEQ ID NO: 3, position 379 in SEQ ID NO: 1 corresponds to amino acid at position 354 in SEQ ID NO: 3, and position 447 in SEQ ID NO: : 1 corresponds to the amino acid at position 422 in SEQ ID NO: 4. In SEQ ID NO: 3, or in an amino acid sequence that is 85%, 90%, or 95% or more identical to SEQ ID NO: 3, a mutation of one or both of these amino acids may be introduced in addition to the mutation in which leucines are replaced by cysteines, the glutamic acid mutation at position 60, and/or the furin recognition site mutation as described above, and further in addition to the mutation at position 189 and/or or at position 190, or at position 42, and/or at position 384, as described above.

[0047]

A specific variant of the mutant RSV F protein of the invention is a mutant RSV F protein comprising the amino acid sequence of any of SEQ ID NO: 6-9. In particular, the following amino acid mutations were included.

[0048]

The amino acid sequence of SEQ ID NO: 6 (RSV_F mutant protein contained in FH_50) is the amino acid sequence in which in SEQ ID NO: 3, the leucine at position 141 of SEQ ID NO: 1 is replaced by cysteine; the leucine at position 373 of SEQ ID NO: 1 is replaced by cysteine; the arginine at position 135 of SEQ ID NO: 1 is replaced by asparagine; the arginine at position 136 of SEQ ID NO: 1 is replaced by asparagine; the threonine at position 189 of SEQ ID NO: 1 is replaced by valine; the serine at position 190 of SEQ ID NO: 1 is replaced by valine; the proline at position 102 of SEQ ID NO: 1 is replaced by alanine; the isoleucine at position 379 of SEQ ID NO: 1 is replaced by valine; and the methionine at position 447 of SEQ ID NO: 1 is replaced by valine. In this case, the amino acid sequence of SEQ ID NO: 6 is actually divided into an amino acid sequence of an F2 polypeptide and an amino acid sequence of an F1 polypeptide with the addition of pep27 (extracellular region). However, in this case, they are represented as a single amino acid sequence in which they are linked together. Thus, when determining the identity of the amino acid sequences described below, comparison is made with an amino acid sequence in which the amino acid sequence of the F2 polypeptide and the amino acid sequence of the F1 polypeptide with the addition of pep27 (extracellular region) of the target protein are linked in that order. The same applies to the sequences presented below.

[0049]

The amino acid sequence of SEQ ID NO: 7 (RSV_F mutant protein contained in FH_81) is the amino acid sequence in which, in SEQ ID NO: 3, the leucine at position 141 of SEQ ID NO: 1 is replaced by cysteine; the leucine at position 373 of SEQ ID NO: 1 is replaced by cysteine; the arginine at position 135 of SEQ ID NO: 1 is replaced by asparagine; the arginine at position 136 of SEQ ID NO: 1 is replaced by asparagine; the threonine at position 189 of SEQ ID NO: 1 is replaced by valine; the serine at position 190 of SEQ ID NO: 1 is replaced by valine; glutamic acid at position 60 of SEQ ID NO: 1 is replaced by methionine; the proline at position 102 of SEQ ID NO: 1 is replaced by alanine; the isoleucine at position 379 of SEQ ID NO: 1 is replaced by valine; and the methionine at position 447 of SEQ ID NO: 1 is replaced by valine.

[0050]

The amino acid sequence of SEQ ID NO: 8 (RSV_F mutant protein contained in FH_82) is the amino acid sequence in which in SEQ ID NO: 3, the leucine at position 141 of SEQ ID NO: 1 is replaced by cysteine; the leucine at position 373 of SEQ ID NO: 1 is replaced by cysteine; the arginine at position 135 of SEQ ID NO: 1 is replaced by asparagine; the arginine at position 136 of SEQ ID NO: 1 is replaced by asparagine; the threonine at position 189 of SEQ ID NO: 1 is replaced by valine; the serine at position 190 of SEQ ID NO: 1 is replaced by valine; the lysine at position 42 of SEQ ID NO: 1 is replaced by arginine; the valine at position 384 of SEQ ID NO: 1 is replaced by threonine; the proline at position 102 of SEQ ID NO: 1 is replaced by alanine; the isoleucine at position 379 of SEQ ID NO: 1 is replaced by valine; and the methionine at position 447 of SEQ ID NO: 1 is replaced by valine.

[0051]

The amino acid sequence of SEQ ID NO: 9 (RSV_F mutant protein contained in FH_85) is the amino acid sequence in which in SEQ ID NO: 3, the leucine at position 141 of SEQ ID NO: 1 is replaced by cysteine; the leucine at position 373 of SEQ ID NO: 1 is replaced by cysteine; the arginine at position 135 of SEQ ID NO: 1 is replaced by asparagine; the arginine at position 136 of SEQ ID NO: 1 is replaced by asparagine; the threonine at position 189 of SEQ ID NO: 1 is replaced by valine; the serine at position 190 of SEQ ID NO: 1 is replaced by valine; glutamic acid at position 60 of SEQ ID NO: 1 is replaced by methionine; the lysine at position 42 of SEQ ID NO: 1 is replaced by arginine; the valine at position 384 of SEQ ID NO: 1 is replaced by threonine; proline at position 102 SEQ ID NO: 1 replaced replaced by alanine; the isoleucine at position 379 of SEQ ID NO: 1 is replaced by valine; and the methionine at position 447 of SEQ ID NO: 1 is replaced by valine.

[0052]

One embodiment of the invention is a mutant RSV F protein having the ability to induce an antibody against the RSV F type pre-protein and containing the amino acid substitutions described above in an amino acid sequence that is 85% or more, preferably 90% or more, and more preferably 95% % or more identical to the amino acid sequence of any of SEQ ID NO: 6-9, where a disulfide bond is formed between the cysteines.

Determination of an amino acid sequence that is identical to an already determined amino acid sequence is the same as described above.

[0053]

Fusion protein

Another embodiment of the present invention is a fusion protein that contains a mutant RSV F protein and at least a multimerization domain.

The fusion protein may contain a polypeptide different from the mutant RSV F protein and a multimerization domain. A polypeptide different from the mutant RSV F protein and the multimerization domain are not particularly limited as long as they do not adversely affect the action of the mutant RSV F protein. However, particular examples of such proteins are motifs such as the thrombin cleavage motif and purification tags such as a His tag or Strep II tag.

[0054]

The multimerization domain is not particularly limited so long as it is a polypeptide required for multimerization of the RSV F mutant protein. However, an example is the foldon domain for trimerization (Yizhi Tao et al., 1997, Structure 5 (6): 789) (Frank S. et al., 2001, Journal of molecular biology 308 (5): 1081).

An example of a foldon domain is the foldon domain obtained from fibritin of bacteriophage T4. The amino acid sequence thereof may be the amino acid sequence of SEQ ID NO: 21.

[0055]

An example of a fusion protein is a fusion protein in which the foldon domain is linked to the C-terminus of a mutant RSV F protein having the amino acid sequence of SEQ ID NO: 6-9.

In particular, examples of such proteins are proteins having the amino acid sequences of SEQ ID NO: 10-13.

In fact, the amino acid sequence of SEQ ID NO: 10-13 is divided into the amino acid sequence of the F2 polypeptide and the amino acid sequence of the F1 polypeptide with the addition of pep27 (extracellular region). However, in this case, they are represented as a single amino acid sequence in which they are linked together.

[0056]

Multimer

Another embodiment of the present invention is a multimer fusion protein. In a multimer, the fusion protein is linked through a multimerization domain, such as a foldon domain. The multimer is preferably a trimer of a fusion protein.

[0057]

Polynucleotide

Another embodiment of the present invention is a polynucleotide encoding a mutant RSV F protein or fusion protein.

The polynucleotide encoding the mutant RSV F protein can be produced as a polynucleotide into which the desired mutation has been introduced using standard genetic engineering techniques based on the polynucleotide sequence (SEQ ID NO: 74) encoding the RSV F protein before introducing the mutation.

In addition, a polynucleotide encoding a fusion protein can also be produced using standard genetic engineering techniques using a polynucleotide encoding a mutant RSV F protein and a polynucleotide encoding a polypeptide different from the mutant RSV F protein.

[0058]

In particular, examples of the polynucleotide sequence to be used for expressing the fusion protein of the invention and producing it in a host include the polynucleotide sequences of SEQ ID NOs: 14-17.

SEQ ID NO: 14 is a polynucleotide sequence designed to express and produce a fusion protein having the amino acid sequence of SEQ ID NO: 10 in a host. However, a signal peptide coding sequence is appended to the 5' end of the fusion protein coding sequence.

SEQ ID NO: 15 is a polynucleotide sequence designed to express and produce a fusion protein having the amino acid sequence of SEQ ID NO: 11 in a host. However, a signal peptide coding sequence is appended to the 5' end of the fusion protein coding sequence.

SEQ ID NO: 16 is a polynucleotide sequence designed to express and produce a fusion protein having the amino acid sequence of SEQ ID NO: 12 in a host. However, a signal peptide coding sequence is appended to the 5' end of the fusion protein coding sequence.

SEQ ID NO: 17 is a polynucleotide sequence designed to express and produce a fusion protein having the amino acid sequence of SEQ ID NO: 13 in a host. However, a signal peptide coding sequence is appended to the 5' end of the fusion protein coding sequence.

[0059]

Any codon can be used as long as it codes for the intended amino acid. For example, there are four types of codons encoding Gly: GGT, GGC, GGA and GGG, and any of them can be used. In addition, each codon may or may not be optimized, but it is preferable that each codon be optimized.

[0060]

A polynucleotide encoding a mutant RSV F protein may be a polynucleotide that hybridizes under stringent conditions to a polynucleotide having a sequence complementary to those polynucleotide sequences, provided that the polynucleotide encoding a mutant RSV F protein will encode a mutant RSV F protein that includes the above specific mutations and has the ability to induce antibody against the RSV type F pre-protein. Here, an example of a stringent condition is the wash condition at a salt concentration corresponding to 68°C, 0.1×SSC, 0.1% SDS after Southern hybridization.

[0061]

Expression cluster

Another embodiment of the present invention is an expression cluster comprising a polynucleotide.

An expression cluster may express a polynucleotide by a specific mechanism, for example, it may include an element necessary for the expression of a promoter sequence, a transcription termination signal sequence, etc., and may be an expression cluster used in standard genetic engineering techniques. The expression cluster may be included, for example, in a recombinant vector. Examples of a recombinant vector are a plasmid vector and a viral vector, and examples of such vectors are a vector that can be expressed in a prokaryotic cell; a vector that can be expressed in a eukaryotic cell; and a vector that can be expressed in a cell derived from a mammal.

[0062]

Host cell

Another embodiment of the present invention is a host cell that includes an expression cassette. Preferably, the host cell is a host cell transformed with a recombinant vector comprising an expression cassette.

The host cell is not particularly limited, provided that the protein encoded by the polynucleotide will be expressed from an expression cluster and that it can be used in standard genetic engineering techniques. In particular, examples of such cells are prokaryotic cells such as Escherichia coli and Bacillus subtilis , eukaryotic cells and mammalian-derived cells.

Introduction of the polynucleotide into a host cell can be accomplished using generally known methods such as the calcium phosphate method, the DEAE-dextran method, the electroporation method, and the lipofection method.

By culturing the host cell under suitable conditions, the mutant RSV F protein or fusion protein can be expressed, and the mutant RSV F protein, fusion protein, or a multimer thereof can be produced. The resulting mutant RSV F protein, fusion protein, or multimer thereof can be purified by generally known methods.

[0063]

Immunogen

Another embodiment of the present invention is an immunogen that includes a mutant RSV F protein, fusion protein, multimer, or a particulate product as described below.

That is, the mutant RSV F protein, fusion protein, multimer or particulate product can be used as an immunogen. The immunogen may include any one, any two, or all of the following proteins, such as a mutant RSV F protein, a fusion protein, a multimer, or a particulate product, so long as the immunogen does not adversely affect the action of the mutant RSV F protein. Among them, a multimer, and especially a trimer, is preferred, and a particulate product is preferred for use as an immunogen. The preparation of the multimer is described above and the particulate product will be described in detail below. The immunogen according to the invention may contain a preservative, excipient, etc., included in the standard immunogen.

[0064]

Particle product

Another embodiment of the present invention is a particulate product of a mutant RSV F protein or multimer.

The particulate product is not particularly limited as long as it is a particle formed by the aggregation of two or more mutant RSV F proteins or multimers and can effectively induce an antibody having high neutralizing ability. However, for example, a particulate product is a particle formed by the aggregation of two or more mutant RSV F proteins or multimers through a particulate domain. In this case, the forms of the product in the form of particles and the particles themselves need not be limited to spherical shapes, and it is sufficient that their aggregation occurs only in a certain order.

That is, it is preferable that the particle-forming domain is also linked to the mutant RSV F protein or fusion protein, and the multimer of the mutant RSV F protein or fusion protein is aggregated through the particle-forming domain to form particles.

Preferably, particle formation is accomplished using a fusion protein to produce a particulate product in which the particle-forming domain is linked to the C-terminus of the mutant RSV F protein or fusion protein. The fusion protein for producing the particulate product will be described below.

[0065]

In the particulate product, the particulate domain is preferably located closer to the I epitope than to the Φ epitope in the steric structure of the mutant RSV F protein or multimer. That is, the preferred product is a particulate product in which the core is formed by aggregation of a particle-forming domain, and the I epitope of the mutant RSV F protein or multimer is present on the inner side, and the Φ epitope is exposed on the outer side.

Thus, the particulate product according to one embodiment of the invention is a particulate product in which two or more mutant RSV F proteins or multimers are aggregated through a particulate domain such that at least the Φ epitope of the RSV F protein is exposed and at least epitope I of the RSV F protein is not so affected. As used herein, the term “epitope I is not affected” does not necessarily mean that epitope I is completely unaffected until the availability of antibodies or various immune cells to epitope I is reduced.

[0066]

In one embodiment of the particulate product of the invention, because the Φ epitope and the I epitope of the mutant RSV F protein are located at two extremes of the steric structure of the protein, the particle-forming domain that forms the core is located closer to the I epitope than to the Φ epitope, and Thus, a structure can be obtained in which epitope I, which is an unnecessary epitope, is disguised as an antigen, and epitope Φ, which is a useful epitope, can easily function as an antigen. As used herein, an unnecessary epitope means an epitope that typically induces an antibody having low ability to neutralize RSV viral infection in humans when a mutant RSV F protein, fusion protein, multimer, or particulate product thereof is used as an immunogen. Specific examples include epitope I. On the other hand, a useful epitope means an epitope that typically induces an antibody that is highly capable of neutralizing RSV virus infection in humans when a mutant RSV F protein, fusion protein, multimer, or particulate product thereof is used as an immunogen . Specific examples of such epitopes include the Φ epitope and the V epitope. The formation of such a structure has the effect that the access of antibodies or various immune cells to the unnecessary epitope is reduced due to the effect of steric hindrance.

[0067]

The particle-forming domain is a polypeptide that converts the mutant RSV F protein or its multimer into particles. Examples are domains that can bind to scaffold particles (eg, scaffold-binding peptides), domains that can aggregate hydrophobically (eg, hydrophobic peptide chains), domains that have the ability to self-associate (eg, self-associating peptides), or the like. .P.

[0068]

In this section, first of all, a description of the variant of particle formation using framework particles is provided. That is, this variant is a mutant RSV F protein or multimer associated with scaffold particles.

The composition of the framework particles has no specific restrictions. However, their examples are proteins having the ability to form particles, lipid bilayers, shells, liposomes, niosomes, virosomes, ferrosomes, gold nanoparticles, resins, silica, polymer micelles, gels, etc.

Examples of proteins having the ability to form particles are viral proteins or variants of viral proteins that form VLPs (virus-like particles) or VPs (viral particles) (see, for example, Japanese Patent Application Laid-Open No. 2004-2313). Thus, for example, one can mention the hepatitis B virus surface antigen protein, modified to remove the original infectivity towards hepatocytes and further modified to present a scaffold molecule, and when expressed in eukaryotic cells, this protein is expressed and accumulated as a membrane protein such as the membrane of the endoplasmic reticulum, and is released as VLP.

More specifically, protein-derived VLPs obtained by inserting the Fc-binding domain (Z-domain) of protein A into the PreS region of hepatitis B virus surface antigen (HBs antigen) may be mentioned as framework particles, and, for example, a bionanocapsule may be used. ZZ (Beacle part number: BCL-DC-002).

Preferably, a molecule (scaffold molecule) that binds to a scaffold particle-binding peptide as a particle-forming domain is immobilized on the surface of the scaffold particle.

Thus, for example, the Fc-binding domain of protein A may be mentioned, and the mutant RSV F protein or a multimer thereof may be linked via a scaffold binding peptide.

[0069]

The bond between the framework molecule and the peptide binding to the framework particle can be either covalent or non-covalent. Examples of making covalent bonds include methods collectively called "click chemistry" (examples include the alkyne (e.g., acetylene)-azide bond, cysteine-maleimide bond, and primary amine NHS ester bond), charge shift bond formation (example is a hydrazide-aldehyde bond), a method of creating a covalent bond depending on the polypeptide (such as a method using SpyTag-SpyCatcher), etc. Examples of non-covalent bonds are ionic bonding, hydrophobic bonding, hydrogen bonding, etc.

In particular, examples are combinations of protein tags and protein tag binding molecules (biotin-avidin, Fc-protein A, GST (glutathione-S-transferase)-glutathione, MBP (maltose-binding protein)-maltose, His-tag- nickel and SBP-streptavidin tag), combination of antibody and antigen, etc.

A preferred combination of a scaffold molecule and a scaffold-binding peptide is a combination of an IgG1 Fc domain (SEQ ID NO: 30) and an Fc binding domain (Z domain) of Protein A. The IgG1 Fc domain may be used for a scaffold molecule, and the Fc binding domain of Protein A can be used for a scaffold binding peptide. The Fc domain of IgG1 can be used as a scaffold binding peptide, and the Fc binding domain of Protein A can be used as a scaffold molecule.

Since the above scaffold molecules or peptides binding to the scaffold particles may be covalently or non-covalently linked to each other, partial fragments thereof may be used or they may be modified.

[0070]

The following describes an option for forming particles using a hydrophobic peptide chain as the particle-forming domain.

In this embodiment, the hydrophobic peptide chain is linked to the mutant RSV F protein or fusion protein, and the mutant RSV F protein or fusion protein forms a particle via the hydrophobic peptide chain.

Hydrophobic peptide chain means a peptide chain that contains a hydrophobic amino acid and undergoes self-assembly to form a particulate product, and which contains 5-50, and preferably 10-30 amino acid residues. Here, examples of highly hydrophobic amino acids include valine, leucine, phenylalanine, isoleucine and the like, and leucine or isoleucine are more preferable. The ratio of hydrophobic amino acids in the hydrophobic peptide chain is preferably 20-60%.

In a hydrophobic peptide chain, it is preferable for the hydrophobic amino acids to be on the surface of the hydrophobic peptide chain. The proportion of hydrophobic amino acids on the surface is preferably 10% or more. However, if the ratio is too high, then these amino acids aggregate inside the cells and are not secreted, and thus the hydrophobic amino acids on the surface are preferably 50% or less, 30% or less, and 20% or less. On the other hand, a higher ratio of hydrophobic amino acids on the inner side results in a stable hydrophobic peptide chain structure and is thus preferred.

In this case, any amino acid can be used, except for a hydrophobic amino acid. However, in particular to enhance the secretory effect, it is preferable that the histidine content is 20% or more.

When a hydrophobic peptide chain is formed into a repeating peptide sequence consisting of 7 amino acids, a helical structure is formed, and the structure described above can be easily obtained, and therefore it is preferable to use it. For example, the hydrophobic peptide chain is preferably a repeating peptide sequence of 7 amino acids, in which the hydrophobic amino acids are located at positions 1, 3 and 5. The number of repeats is, for example, 2-5, and preferably 2-3.

Preferable examples of the hydrophobic peptide chain include CC02, CC03, CC07 and CC08 (in that order, SEQ ID NO: 40, 41, 42 and 43), and among them, CC07 (SEQ ID NO: 42) is particularly preferred.

[0071]

The following describes an embodiment of particle formation using a self-associating protein domain as the particle-forming domain.

In this embodiment of the invention, the self-associating protein domain is linked to a mutant RSV F protein or fusion protein, and the mutant RSV F protein or fusion protein forms a particle through the self-associating protein domain.

Examples of self-associating proteins are hepatitis B virus core antigen (HBcAg), human papillomavirus capsid protein (HPV L1), hepatitis E virus capsid protein (HEV capsid), bacteriophage Qβ envelope protein, Norwalk virus capsid (Norovirus, NoV), parvovirus B19 capsid , alfalfa mosaic virus, feritin, encapsulin, etc. The self-associating protein may be a self-associating partial fragment or variant.

In the case where HBcAg is used, when binding to the RSV F protein or a multimer according to the invention, it is preferable that the RSV F protein or a multimer thereof binds to a site distant from the association surface for HbcAg self-association and on the outside of the self-associating proteins. In addition, when performing such linking, it is preferable to insert a linker between the α3 domain helix and the α4 domain helix to obtain the original steric structure of HBcAg. The linker is, for example, a polypeptide containing 10-30 amino acids, and, for example, a GS linker. An example of a sequence in which a FLAG tag is associated with such an HBcAg variant is SEQ ID NO: 54.

[0072]

<Fusion protein to produce particulate product>

A particulate product fusion protein is a protein used to produce the above particulate product and contains a particulate domain, such as a particle binding scaffold peptide, a hydrophobic peptide chain, or a self-associating protein domain described above, which linked to the C-terminus of a mutant RSV F protein of the invention or to the C-terminus of a fusion protein.

The fusion protein of the invention used to produce a particulate product is aggregated through a particulate domain to form a particulate product.

If the distance between the C-termini of the fusion proteins forming a multimer according to the invention is changed due to binding to the particle-forming domain by inserting a linker between the particle-forming domain and the C-termini (the multimerization domain), then the distance between the C- ends can be reduced. The linker is not particularly limited, but preferably it is a peptide containing 5-20 amino acids, and more preferably, a peptide containing 5-10 amino acids. The sequence is not particularly limited as long as it does not inhibit the effect of the fusion protein on the vaccine, but, for example, could be a GS linker consisting of glycine and serine.

The linker may be located between the multimerization domain and the particle formation domain of the mutant RSV F protein or fusion protein, and tag sequences may be included before and after the linker. Tag sequences have no specific restrictions. However, it is preferable to use tag sequences that are commonly used for protein purification. Examples include a FLAG tag (SEQ ID NO: 28), a His tag (SEQ ID NO: 25), a Strep II tag (SEQ ID NO: 26), and the like.

[0073]

The fusion protein used to produce a particulate product can be produced by linking, in frame, DNA encoding the particle-forming domain, and optionally DNA encoding a linker or tag, to DNA encoding the mutant F protein RSV or fusion protein and its expression in the host. The expression and purification method can be carried out by the same method as the expression and purification method of the RSV F mutant protein or fusion protein.

[0074]

Specific examples of a fusion protein used to produce a particulate product include one in which the scaffold binding peptide is linked to the mutant RSV F protein (precursor protein sequences thereof: SEQ ID NOs: 31-39, and the polynucleotides encoding the same sequences: SEQ ID NO: 57-65); a variant in which the hydrophobic peptide chain is associated with a mutant RSV F protein (sequences of its precursor proteins: SEQ ID NO: 44-49, and polynucleotide sequences encoding it: SEQ ID NO: 66-71), and a variant in which the self-associating domain protein is associated with the mutant RSV F protein (sequences of its precursor proteins: SEQ ID NO: 55-56, or polynucleotide sequences encoding it: SEQ ID NO: 72-73).

The fusion protein used to produce the particulate product may contain tag sequences such as a FLAG tag (DYKDDDDK: SEQ ID NO: 28) and a tag (GGSDYKDDDDK: SEQ ID NO: 29), where 3 amino acids (GGS) are attached to the tag FLAG.

[0075]

Method for obtaining a product in the form of particles

The particulate product can be produced by forming particles using the fusion protein obtained above to produce a particulate product, followed by purification of the product if necessary.

When using scaffold particles, the particle formation step can be accomplished by mixing the fusion proteins to produce particulate products with the scaffold particles, and then removing mutant RSV F proteins or its products that do not form particles and purifying the particulate products. For example, HBsAg VLP (Beacle catalog number: BCL-DC-002) can be used as framework particles.

When using fusion proteins to produce particulate products containing hydrophobic peptide chains, the particle formation step can be carried out by coupling the fusion proteins to produce particulate products through the hydrophobic peptide chains to form particulate products, and then removing the mutant RSV F proteins or non-particulate products thereof, and purification of particulate products.

When using fusion proteins to produce particulate products containing self-associating protein domains, the particle formation step can be accomplished by linking the fusion proteins to produce particulate products through the self-associating protein domains to form particulate products, and then removing the mutant RSV F proteins or non-particulate products thereof, and purification of particulate products.

[0076]

In the particulate products of the invention, it is desirable that there are, for example, 2 or more, preferably 10 or more, and more preferably 20 or more bound trimers per particulate product.

In addition, in the particulate products of the invention, it is desirable that per 1 nm ² surface area of the particulate product there are, for example, 0.0001 or more, preferably 0.0005 or more, and more preferably 0.001 or more bound trimers. Here, the surface area of a particulate product means the surface area if the particulate product is assumed to be a sphere, the diameter of which is the theoretical diameter measured by dynamic light scattering. The number of particles per unit surface area of the particulate product can be calculated, for example, using a method in which the number of particles per unit of particulate product is divided by the surface area of the particulate product.

When preparing particulate products and selecting products that have a low binding affinity for the I epitope and a high binding affinity for the Φ epitope, one skilled in the art can select the optimal number of particles per particulate product or the optimal number of particles per 1 nm ² area surface of the product in the form of particles.

[0077]

In the present invention, the RSV F protein or multimer used to form particles is not limited to the mutant RSV F protein or multimer according to the invention and is not particularly limited provided that it can be used as an RSV vaccine, and a mutant can also be used an RSV F protein different from the mutant RSV F protein of the invention, or a multimer thereof. For example, proteins described in International Publication No. 2017/172890 or International Publication No. 2017/109629 can be used. That is, the particle formation technology of the invention can generally be used to form particles of RSV F proteins or multimers thereof. The particulate products of the invention are particulate products of RSV F proteins or multimers thereof and include particulate products formed by the aggregation of two or more RSV F proteins or multimers thereof through particulate domains. As the particle-forming domains and the particle-forming method, the particle-forming domains and particle-forming methods described above for particle formation of mutant RSV F proteins or multimers thereof can be used.

[0078]

Pharmaceutical composition

Another embodiment of the present invention is a pharmaceutical composition comprising an expression cluster or immunogen as an active ingredient.

The expression cluster or immunogen may be a pharmaceutical composition for the prevention or treatment of RS virus infection.

Another embodiment of the present invention is an RSV vaccine that includes an expression cluster or immunogen. This option is also an option in which the pharmaceutical composition according to the above option is an RSV vaccine.

[0079]

The pharmaceutical composition (including the RSV vaccine, also referred to below) is not particularly limited in terms of the route of administration and can be administered to a mammal either by parenteral administration (for example, intradermal, intramuscular, subcutaneous, transdermal and transmucosal administration) or orally introduction. Examples of mammals include humans, mice, rats, rabbits, dogs, cats, cows, pigs, monkeys, chimpanzees and the like, and preferably humans.

[0080]

The dosage form of the pharmaceutical composition and the method for its preparation are well known to those skilled in the art, and the pharmaceutical composition can be prepared by mixing the expression cluster or immunogen with a pharmaceutically acceptable carrier or the like.

[0081]

Examples of a pharmaceutically acceptable carrier include an excipient, a lubricant, a binder and a disintegrating agent in a solid composition or a solvent, a solubilizing agent, a suspending agent, an isotonicizing agent, a buffering agent, an emollient, and the like. in a liquid composition. In addition, if necessary, additives such as a conventional antiseptic, antioxidant, colorant, sweetener, adsorbent and wetting agent can be suitably used in appropriate amounts.

[0082]

The pharmaceutical composition may include an adjuvant. Examples of the adjuvant are gel, bacterial cell toxins, oil emulsion, polymer nanoparticles, synthetic adjuvant, cytokine, and the like.

Examples of gels include aluminum hydroxide, aluminum phosphate, calcium phosphate and the like.

Examples of bacterial cell toxins include CpG, cholera toxin, thermophilic Escherichia coli toxin, pertussis toxin, muramyl dipeptide (MDP), and the like.

Examples of oil emulsions are Freund's incomplete adjuvant, MF59, SAF, etc.

Examples of polymeric nanoparticles are immunostimulatory complexes, liposomes, biodegradable microspheres, saponin-derived QS-21, and the like.

Examples of synthetic adjuvants are nonionic block copolymers, muramyl peptide analogs, polyphosphazene, synthetic polynucleotides, and the like.

Examples of cytokines are IFN-γ, IL-2, IL-12 and the like.

[0083]

Examples of dosage forms for parenteral administration are injection preparations (such as drip injection, intravenous injection, intramuscular injection, subcutaneous injection, intradermal injection, intracerebral injection and intraspinal preparation), topical preparations (such as ointment, poultice and lotions), inhalation suppositories, ophthalmic agents, eye ointments, nasal drops, ear drops, liposomal agents, etc.

Examples of dosage forms for oral administration are solid or liquid dosage forms, such as tablets, coated tablets, pills, granules, granules, powders, capsules, syrups, emulsions, suspensions, troches, and the like.

The pharmaceutical composition according to the invention is preferably a liquid dosage form, and more preferably an injection.

[0084]

The effective dose of a pharmaceutical composition can be determined, for example, by a physician based on various factors such as route of administration, type of disease, severity of symptoms, age, sex and body weight of the patient, severity of the disease, pharmacological data such as pharmacokinetics and toxicological features, regardless of whether a drug delivery system is used or not, and whether the pharmaceutical composition is administered as part of a combination with other drugs or not.

Examples

[0085]

Below is a description of the Examples. However, none of these examples should be interpreted as limiting the scope of the invention.

[0086]

Example 1

The formation of a covalent bond (disulfide bond) using cysteines helps stabilize the protein structure. Previous studies have reported various disulfide mutations that stabilize the pre-F structure in the RSV F protein (hereinafter may be referred to as the “F protein”) (International Publication No. 2017/172890 and International Publication No. 2017/109629). However, since the amino acid included in the secondary structure is replaced by a cysteine or forms a disulfide in a distant part, there is a high risk that the resulting mutant may have a structure different from the natural structure (the prefusion structure that is believed to have the natural F protein during its expression). Therefore, it was decided to stabilize the structure by replacing an amino acid pair with cysteines, which has no secondary structure and has a distance between S atoms of less than 5 Å when replaced with cysteines. In calculating the distance, a published structural model (PDB accession number: 4MMS) was used and two arbitrary amino acids were replaced with cysteines to obtain a conformation having an energetically stable N-Ca-Cb-S dihedral angle, and the shortest distance between S atoms was calculated (Table 1). As a result, mutation sites of the mutant RSV F protein (which may be referred to herein as "mutant protein") contained in the FH_08 and FH_09 antigens, which will be described later, have been listed as candidates. These two candidates do not have a polar functional group on the amino acid side chain before the substitution at the mutation site, and are thus considered superior also in that there is no risk of structural instability due to loss of polar group interaction (hydrogen bond or the like) .) present in naturally occurring protein F. “L141C” in Table 1 means that the leucine at position 141 of amino acid sequence SEQ ID NO: 1 has been replaced by cysteine. Here and further in the Table the same notations are used. The native F antigen precursor protein (SEQ ID NO: 4) is a polypeptide, wherein said polypeptide (amino acid numbers 1-513 SEQ ID NO: 1) obtained by removing the transmembrane region and intracellular region of the F1 polypeptide from the native F0 protein of strain A2 , a foldon domain (SEQ ID NO: 21) and a sequence (SEQ ID NO: 22) including a thrombin recognition sequence, a His tag and a Strep tag II are sequentially attached to the C-terminal side of a polypeptide having the mutations P102A, I379V and M447V . In this example, all mutants were created from the native F antigen precursor protein and thus have the mutations P102A, I379V, and M447V. The native F precursor protein is processed during expression, resulting in the removal of the signal peptide and the pep27 region.

[0087]

Table 1

Antigen name Mutation content Result of structural analysis Distance between atoms S (Å) Secondary structure Native F None - FH_08 L141С, L373С 4.35 Absent FH_09 L142C, L373C 4.72 Absent

[0088]

Native F antigen is produced by expressing a polynucleotide (SEQ ID NO: 5) encoding a precursor protein (SEQ ID NO: 4) of native F antigen using a mammalian cell as the host. The native F antigen forms a quaternary structure after the native F antigen precursor protein (SEQ ID NO: 4) is processed during expression. As a result of processing, the signal peptide and pep27 region are removed, and the remaining sequence of the F2 polypeptide and the sequence including the F1 polypeptide (extracellular region), foldon domain, thrombin recognition sequence, His tag and Strep II tag are linked by a disulfide bond, and in addition , the homotrimer is expected to form due to the binding force mainly generated by the foldon domain.

A specific method for producing native F antigen is shown below. The above polynucleotide (SEQ ID NO: 5) was inserted into the pcDNA3.4 vector (Thermo Fisher Scientific, Inc.) using standard genetic engineering techniques. The insertion of the target DNA sequence was confirmed by base sequence analysis, and a mammalian cell expression vector expressing the native F antigen precursor protein was constructed. The vector for introduction into the cell was purified by a standard plasmid vector purification method.

[0089]

Expression of native F antigen was accomplished by expression of the secreted protein in mammalian cells using the Expi293 expression system (Thermo Fisher Scientific, Inc.). The purified vector was introduced into an Expi293 cell using the Expi Fectamin 293 reagent, which is a gene transfer reagent from the same kit, and then the encoded native F antigen was secreted and expressed by culturing for approximately 5 days, and only the supernatant component was obtained by centrifugation and using a filter with a hole diameter of 0.22 microns.

[0090]

Purification of native F antigen was performed using affinity chromatography on a Ni-Sepharose high performance column (GE Healthcare, Inc.). The Ni carrier was reacted with the above component of the culture supernatant, diluted approximately 3 times with binding buffer (20 mmol/L Tris-HCl, pH 7.4, 500 mmol/L NaCl, 20 mmol/L imidazole) for 24 hours, and then it was applied to elution buffer (20 mmol/L Tris-HCl, pH 7.4, 500 mmol/L NaCl, 500 mmol/L imidazole) to elute each antigen. Subsequently, solvent exchange with PBS (pH 7.4) was carried out using an ultrafiltration membrane, and the product was used as native F antigen.

[0091]

The polynucleotide encoding the FH_08 antigen precursor protein, where the double mutation L141C and L373C was introduced into the native F antigen precursor protein, was prepared as described above. Next, in the same manner as described above, a polynucleotide encoding the precursor protein of the FH_09 antigen was obtained, where the double mutation L142C and L373C was introduced into the precursor protein of the native F antigen. These proteins were expressed and purified using the method described above for obtaining the FH_08 antigens and FH_09.

[0092]

The binding capacity of each antigen to each antibody was assessed using Octet QK384 (ForteBio, Inc.) under PBS (pH 7.4), 25°C, and 1000 rpm. Each antibody was coupled to the sensor chip by applying each antibody solution for 180 seconds to a Protein A sensor chip (ForteBio, Inc.) equilibrated with PBS (pH 7.4), and increasing the detection value if each antigen solution was applied sequentially for 180 seconds, was defined as the value of the binding signal of each antigen to each antibody. Antibody D25, which can specifically detect the Φ epitope, was used as an antibody applied to the sensor chip. The magnitude of the binding signal was normalized by determining the ratio to the magnitude of the binding signal using palivizumab (Synagis intramuscular injection, AbbVie, Inc.), which binds independently of the conformational change of the F antigen.

[0093]

Using the above procedure, a result was obtained indicating that the antigens FH_08 and FH_09, containing mutant proteins in which cysteine point mutations were introduced at two sites to form a disulfide bond, significantly improved the storage stability of the Φ epitope compared with the native antigen F containing the F protein before the introduction of mutations (Fig. 1).

[0094]

Example 2

There is a report that the native F protein is first cleaved at the first recognition site by furin to form a monomeric structure, and then cleaved at the second recognition site by furin to remove the pep27 region, thereby promoting trimerization (Anders Krarup et al., 2015, Nature communications 6:8143). However, since the pep27 region is highly conserved in sequence length and amino acid type among virus strains isolated from a natural source, it is believed that the pep27 region may have an indispensable influence on the formation of the steric structure of the antigen. Therefore, a mutation was introduced into the second furin recognition site to inhibit cleavage of the pep27 region, and the effect of the mutation on trimer formation was examined. The mutation contents of the antigen precursor proteins obtained as described below have been systematized in Table 2. “FH_08+R135N, R136N” in Table 2 means that in the antigen precursor protein FH_08, arginine is at position 135 in the amino acid sequence SEQ ID NO: 1 was replaced by asparagine, and the arginine at position 136 was replaced by asparagine. "FH_09+R135N, R136N" means that in the FH_09 antigen precursor protein, arginine at position 135 in the amino acid sequence of SEQ ID NO: 1 was replaced by asparagine, and arginine at position 136 was replaced by asparagine. In the FH23-25 antigens, the pep27 (p27) sequence in the FH08 antigen precursor protein was replaced by various GS linkers. GSGSGS (SEQ ID NO: 18), (GGGGS) ₂ (SEQ ID NO: 19), (GGGGS) ₃ (SEQ ID NO: 20) were used as GS linkers.

[0095]

table 2

Antigen name Description of mutations FH_08 L141С, L373С FH_09 L142C, L373C FH_23 Replacement of p27 FH_08 sequence with GSGSGS (SEQ ID NO: 18) FH_24 Replacement of p27 FH_08 sequence with (GGGGS) ₂ (SEQ ID NO: 19) FH_25 Replacement of p27 FH_08 sequence with (GGGGS) ₃ (SEQ ID NO: 20) FH_21 FH_08+R135N, R136N FH_22 FH_09+R135N, R136N

[0096]

The polynucleotide encoding each of the antigen precursor proteins was inserted into the pcDNA3.4 vector (Thermo Fisher Scientific, Inc.) using standard genetic engineering techniques. The insertion of the target DNA sequence was confirmed by base sequence analysis, and each of the mammalian cell expression vectors expressing the antigen precursor proteins was constructed. The vector for introduction into the cell was purified using a standard plasmid vector purification method.

[0097]

Expression and purification of each antigen was carried out using the procedure described in Example 1.

The binding ability of each antigen to each antibody was also assessed using the procedure described in Example 1. However, AM14 antibody, capable of detecting pre-F trimer formation, was added as an antibody to be applied to the sensor chip. The magnitude of the binding signal of antibody D25 and antibody AM14 was normalized by determining the ratio relative to the magnitude of the binding signal when using the Synagis antibody (AbbVie, Inc., Synagis intramuscular injection), which is able to bind independently of the conformational change of the F antigen.

[0098]

Using the procedure described above, a result was obtained indicating that the antigens FH_21, FH_24 and FH_25, containing mutant proteins, each of which had a mutation added to the second furin recognition site, improved the formation of trimers while maintaining the stability of the Φ epitope during storage compared with the FH_08 antigen before introducing the mutation. Among them, the improvement in trimer formation was most noticeable in the FH_21 antigen, which is located closer to the pep27 region of the native F antigen compared with antigens (FH_24 and FH_25) in which the pep27 region was replaced by an artificial linker (Figs. 2 and 3).

[0099]

Example 3

The amino acids at positions 189 and 190 of the native F antigen precursor protein are threonine and serine and are hydrophilic amino acids because each has a hydroxyl group on the side chain. However, amino acids located sterically closer to the amino acids at positions 189-190 are isoleucine at position 57, isoleucine at position 167, leucine at position 171, valine at position 179 and leucine at position 260, etc. However, since they are all hydrophobic amino acids, it is believed that structural stability may be compromised. Therefore, the possibility of structural stabilization and stimulation of increased expression levels is being considered by replacing amino acids in positions 189-190 with hydrophobic amino acids (valine, leucine, isoleucine). Descriptions of mutations in the antigen precursor proteins obtained as described below are systematized in Table 3. “FH_21+T189I” in Table 3 means that in the antigen precursor protein FH_21, the threonine at position 189 in the amino acid sequence SEQ ID NO: 1 is replaced by isoleucine. “FH_21+T189V” means that in the FH_21 antigen precursor protein, threonine at position 189 in the amino acid sequence SEQ ID NO: 1 is replaced by valine. “FH_21+T189I, S190V” means that in the FH_21 antigen precursor protein, threonine at position 189 in the amino acid sequence SEQ ID NO: 1 is replaced by isoleucine, and serine at position 190 is replaced by valine. “FH_21+T189L, S190V” means that in the FH_21 antigen precursor protein, threonine at position 189 in the amino acid sequence SEQ ID NO: 1 is replaced by leucine, and serine at position 190 is replaced by valine. "FH_21+T189V, S190V" means that in the FH_21 antigen precursor protein, threonine at position 189 in the amino acid sequence SEQ ID NO: 1 is replaced by valine, and serine at position 190 is replaced by valine. “FH_50+K42R, V384T” means that in the FH_50 antigen precursor protein, lysine at position 42 in the amino acid sequence SEQ ID NO: 1 is replaced by arginine, and valine at position 384 is replaced by threonine. In addition, point mutations found in natural virus isolates were removed and each individual mutation was examined. The effect of increasing the expression level of two point mutations (K42R, V384T) was confirmed, and thus the study was carried out by introducing such a mutation.

[0100]

Table 3

Antigen name Description of mutations FH_21 L141C, L373C, R135N, R136N FH_55 FH_21+T189I FH_53 FH_21+T189V FH_52 FH_21+T189I, S190V FH_51 FH_21+T189L, S190V FH_50 FH_21+T189V, S190V FH_82 FH_50+K42R, V384T

[0101]

The polynucleotide encoding each of the antigen precursor proteins was inserted into the pcDNA3.4 vector (Thermo Fisher Scientific, Inc.) using standard genetic engineering techniques. The insertion of the target DNA sequence was confirmed by base sequence analysis, and mammalian cell expression vectors expressing antigen precursor proteins were each constructed. The vector for introduction into the cell was purified using a standard plasmid vector purification method.

[0102]

Expression and purification of each of the antigens was carried out using the procedure described in Example 2.

The expression level of each antigen was assessed by measuring the absorbance level using a standard spectrophotometer. The expression level y (y=A280xM/εxVp/Vc) per volume of expression culture was calculated from the absorption (at a wavelength of 280 nm) A280 of the purified antigen solution, from the molar absorption coefficient ε and from the molecular weight M, which were estimated based on information about the primary amino acid sequence, by volume of Vp antigen solution after purification and by volume of Vc expression culture.

[0103]

The binding ability of each antigen to antibody D25 and antibody AM14 was assessed using the procedure described in Example 2.

Using the above procedure, a result was obtained showing that the antigens FH_50, FH_51, FH_52, FH_53 and FH_55, each of which had a mutation, namely, replacement with hydrophobic amino acids (V, I, L) at positions 189-190, significantly increased the expression level while maintaining the stability of the F epitope during storage and trimer formation compared to the FH_21 antigen before the introduction of the mutation. In addition, a result was obtained showing that the FH_82 antigen, to which two point mutations (K42R, V384T) were added, derived from natural mutations that can occur in natural viruses, also significantly increased the level of expression while maintaining the stability of the F epitope during storage and trimer formation (Fig. 4-6).

[0104]

Example 4

The amino acid at position 60 of the native F precursor protein is glutamic acid and is an acidic amino acid and is well known to be negatively charged under conditions where the solvent is neutral. This amino acid is sterically adjacent to aspartic acid at position 194, which is also negatively charged under neutral conditions, and therefore there is potential for structural instability due to the proximity of negative charges. Consequently, a mutation was introduced into the amino acid sequence of the native F precursor protein, namely, the replacement of the amino acid at position 60 with an amino acid that is not an acidic amino acid. However, when replacing the amino acid at position 60 with asparagine, to prevent the amino acid at positions 60-62 from converting into the well-known N-type sugar chain binding motif (Asn-X-Ser/Thr), the serine at position 62 was simultaneously replaced with alanine. Descriptions of mutations in antigen precursor proteins obtained as described below are summarized in Table 4.

[0105]

Table 4

Antigen name Antigen name Native F None FH_124 E60R FH_125 E60M FH_126 E60F FH_57 E60A FH_58 E60H FH_59 E60I FH_60 E60L FH_61 E60N, S62A FH_62 E60Q FH_63 E60S FH_64 E60T FH_65 E60V FH_66 E60W FH_67 E60Y

[0106]

The polynucleotide encoding each of the antigen precursor proteins was inserted into the pcDNA3.4 vector (Thermo Fisher Scientific, Inc.) using standard genetic engineering techniques. The insertion of the target DNA sequence was confirmed by base sequence analysis, and mammalian cell expression vectors expressing antigen precursor proteins were each constructed. The vector for introduction into the cell was purified using a standard plasmid vector purification method.

[0107]

Expression and purification of each of the antigens was carried out using the procedure described in Example 2.

The binding ability of each antigen to antibody D25 and antibody AM14 was assessed using the procedure described in Example 2.

Using the above procedure, a result was obtained showing that the storage stability of the Φ epitope and trimer formation were improved when substitutions of M, F, L, S and T were introduced at position 60 (FIGS. 7 and 8).

[0108]

Example 5. Comparison with predecessor

One of the most widely known mutations introduced as a method of stabilizing the F type pre-protein of the F antigen is the DS-Cav1 mutation (S155C, S290C, S190F, V207L) (Jason S. McLellan et al., 2013, Science 342:592) . Thus, the antigens presented in Table 5, in which beneficial mutations were further introduced into the desired FH_50 antigen and the FH_50 antigen described in the above examples, were compared with the F_DS-Cav1 antigen, into which the DS-Cav1 mutation was introduced. In addition, the ΔFP furin _wt F _ecto antigen was also obtained (Kurt A. Swanson et al., 2014, Journal of Virology, 88 (20): 11802), which is known to have a post-F trimeric structure, and this antigen was used to evaluate antigens. Descriptions of the mutations in the antigen precursor proteins obtained as described below are summarized in Table 5. Each of the FH_81-85 antigen precursor proteins was generated by introducing the E60M, K42R+V384T, or E60M+K42R+V384T mutation into the FH_50 antigen precursor protein.

[0109]

Table 5

Antigen name Description of mutations F_DS-Cav1 S155C, S290C, S190F, V207L FH_50 L141C, L373C, R135N, R136N, T189V, S190V FH_81 FH_50+E60M FH_82 FH_50+K42R, V384T FH_85 FH_50+E60M, K42R, V384T

[0110]

The polynucleotide encoding each of the antigen precursor proteins was inserted into the pcDNA3.4 vector (Thermo Fisher Scientific, Inc.) using standard genetic engineering techniques. The F_DS-Cav1 antigen precursor protein is the amino acid sequence consisting of SEQ ID NO: 23, and the ΔFP furin _wt F _ecto precursor protein is the amino acid sequence consisting of SEQ ID NO: 24. The insertion of the target DNA sequence was confirmed with using base sequence analysis, and each of the mammalian cell expression vectors expressing antigen precursor proteins was constructed. The vector for introduction into the cell was purified using a standard plasmid vector purification method.

[0111]

Expression and purification of each of the antigens was carried out using the procedure described in Example 2.

The expression level of each antigen was assessed using the procedure described in Example 3.

[0112]

Antibody binding test

The binding ability of each antigen to each antibody was assessed using the procedure described in Example 2. However, antibody 131-2A, capable of recognizing epitope I, was added as an antibody to be applied to the sensor chip. Signal magnitude of binding of antibody D25 and antibody AM14 and antibody 131 -2A was normalized by determining its ratio to the binding signal value using the Synagis antibody (AbbVie, Inc., Synagis intramuscular injection), which is capable of binding regardless of the conformational change of the F antigen.

[0113]

Using the above procedure, a result was obtained showing that for the FH_50, FH_81, FH_82 and FH_85 antigens, compared with the F_DS-Cav1 antigen described in the literature, although there was no significant change in stability during storage of the Φ epitope and trimer formation, the I epitope, which , as is known, induces antibodies with a rather low ability to neutralize viral infection, practically undetectable (Fig. 9-11). This suggests that although the vaccine is associated with a risk of exacerbation of symptoms due to immune cell activation that contributes little to neutralizing the virus, this risk is reduced with the introduction of the newly discovered mutations compared with the risk with the introduction of the DS-Cav1 mutation.

[0114]

Test for adsorption of virus-neutralizing antibodies in human serum

As a result of the above procedure, it was shown that there is a possibility that the antigens FH_50, FH_81, FH_82, FH_85 have a suppressive effect in inducing immunity, which does not contribute to the neutralization of the virus. To study this effect in more detail using sera from healthy adults, we tested which types of antibodies in the sera of healthy adults the antigens FH_50, FH_81, FH_82, FH_85 and F_DS-Cav1 bind to.

[0115]

Sera from three healthy adults were subjected to antigen adsorption using magnetic Dynabeads followed by His-tag extraction to produce the desired product (Thermo Fisher Scientific, 10103D). First, 20 μg of F_DS-Cav1, FH_50, FH_81, FH_82, or FH_85 antigen was applied and stimulated to bind to beads in a 45 μl solution washed twice with PBS (pH 7.4) and then blocked with bovine serum albumin. Next, 100 μl of serum from each healthy adult was diluted 10-fold with PBS (pH 7.4), and then beads were added and the mixture was incubated (4°C, 1 hour, with shaking), after which antibodies bound to the antigens were removed by removing balls.

[0116]

An ELISA assay in which the ΔFP furin _{wt Fecto} antigen was immobilized was performed to confirm the level of binding of the antibody (an antibody expected to have a relatively low ability to neutralize viral infection) to post-F. Carbonate-bicarbonate buffer (Sigma-Aldrich, C3041-50CAP) was used as the antigen binding buffer, and TBS (TaKaRa, T9142) was used for sample dilution and plate washing. 2.0 pmol of ΔFP furin _wt F _ecto antigen was added to half the wells of a 96-well plate for direct immobilization and blocking with bovine serum albumin. A series of 4-fold dilutions were prepared by appropriately adding buffer to healthy adult serum or to healthy adult serum after the antigen adsorption procedure, and then added to the plate and incubated (at room temperature, 1 hour, under stationary conditions). Subsequently, the diluted serum was removed and coupled with a peroxidase-conjugated anti-human IgG antibody (Jackson Immuno Research, 709-035-149) and then developed with a standard peroxidase detection reagent, after which the intensity of the color development was determined by measuring absorption on a standard tablet reader. The relationship between the rate of serum dilution and absorbance was calculated by piecewise linear interpolation between measurement points, and the reciprocal of the degree of dilution at which absorbance was 0.2 was defined as the titer of antibody binding to post-F and the relative magnitude of the decrease in serum antibody titer after adsorption procedure, if the serum antibody titer before the adsorption procedure was 100%, was defined as the adsorption rate (Fig. 12).

[0117]

To confirm the extent to which antibody capable of neutralizing viral infection was removed by the adsorption procedure, infection assessment was performed using RS virus (ATCC, VR-1540) and Vero cells (ATCC, CCL-81). A series of 5-fold dilutions were prepared by appropriately adding PBS (pH 7.4) to healthy adult serum or to healthy adult serum after an antigen adsorption procedure, and then mixed with RS virus and incubated (at room temperature, 30 minutes, in stationary conditions). then 4 x 10 ⁴ cells were seeded into each well of a 96-well plate, added to Vero cells, cultured for 1 day, and incubated (37°C, humidified, 2 hours, stationary). After this, the virus solution was removed, followed by a culture medium including DMEM and high glucose (Thermo Fisher Scientific, 11965), into which a sodium pyruvate solution (Nakarai, 06977-34) and a mixed stock solution of antibiotics and antifungals (Nakarai, 09366) -44) were added at 1% (v/v), added at a concentration of 100 μl per well, and further incubated (37°C, humidified, 16 h, stationary). After this, the cells were immobilized with paraformaldehyde, and for cells permeabilized with Triton X-100 (Nakarai, 35501-15), the nuclei of infected cells and all cells were stained using antibodies against RSV, fusion proteins, all strains type A, B, clone 133- 1H, Alexa Fluor 488 (Merck, MAB8262X) and Heochst33258 (Dojindo, 343-07961), and the number of infected cell nuclei was counted on an IN Cell Imaging Scanner (GE Healthcare) to determine the number of virus-infected cells. The relationship between the degree of dilution X of each serum and the number of infections Y was determined by constructing a sigmoid curve (y=Ymax/(1+(x/IC50)C)) from the experimental results (obtained by calculating errors using least squares methods), and as titer neutralizing viral infection, the reciprocal of the IC50 value was used. If the titer neutralizing viral infection in the serum before the adsorption procedure was taken as 100%, then the relative magnitude of the decrease in the titer neutralizing viral infection after the adsorption procedure was taken as the adsorption rate (Fig. 13).

[0118]

Using the above procedure, it was shown that, compared with the F_DS-Cav1 antigen described in the literature, the FH_50, FH_81, FH_82 and FH_85 antigens have a low ability to bind to the post-F recognition antibody present in the serum of a healthy adult, and only approximately half of the antibodies could be bound during the adsorption process. However, it has also been shown that almost all antibodies that contribute to the ability to neutralize viral infection bind similarly to F_DS-Cav1, and the antibodies remaining after the adsorption procedure have almost no ability to neutralize viral infection. The above results suggest that compared to the introduction of the DS-Cav1 mutation, the newly discovered mutation has a lesser ability to bind to an antibody that is ineffective in neutralizing viral infection, and using this mutation as a vaccine antigen will reduce the risk of inducing an antibody that is does not help protect against viral infection.

[0119]

Confirmation of antibodies induced by murine immunity

It was hypothesized that by using the procedure described above, the mutant protein would have an immune suppressive effect that does not help neutralize the virus. To study this effect in more detail, we analyzed antibodies induced by immunizing mice with FH_50, FH_81, FH_82, FH_85 or F_DS-Cav1 antigen.

[0120]

Mice immune sera were obtained by immunizing BALB/cAnNCrlCrlj (Japanese Charles River) (7-week-old females) with mutant proteins. A solution prepared by thoroughly mixing FH_50, FH_81, FH_82, FH_85 or F_DS-Cav1 antigen (antigen concentration: 0.80 mg/ml) dissolved in PBS (pH 7.4) and aluminum hydroxide (InvivoGen, vac-alu- 250), equal amounts were injected intramuscularly in an amount of 50 μl into the hind thighs of mice on the 0th day of immunization and on the 21st day of immunization (5 mice per group). Blood was obtained from the posterior vena cava on day 42 of immunization, and the resulting blood was incubated (at room temperature, approximately 2 hours, under stationary conditions), and then the serum was collected by centrifugation and inactivated (55°C, 30 minutes) to prepare the immune mouse serum.

[0121]

To confirm the ratio at which antibodies are induced that bind to postfusion conformations, each mouse immune serum was subjected to antigen adsorption using magnetic Dynabeads, followed by extraction of the His tag to obtain the desired product (Thermo Fisher Scientific, 10103D). First, 20 μg of ΔFP furin _{wt Fecto} antigen was applied and stimulated to bind to beads in a 45 μl solution washed twice with PBS (pH 7.4) and then blocked with bovine serum albumin. Next, 50 μl of each immune mouse serum was diluted 20-fold with PBS (pH 7.4), and then beads were added and the mixture was incubated (4°C, 1 hour, with shaking), after which antibodies bound to the antigens were removed by removing the beads .

[0122]

An ELISA assay was then performed in which the F_DS-Cav1 antigen was immobilized, and the degree of reduction in antibody binding to the F antigen was assessed by an antigen adsorption procedure. Carbonate-bicarbonate buffer (Sigma-Aldrich, C3041-50CAP) was used as the antigen binding buffer, and TBS (TaKaRa, T9142) was used for sample dilution and plate washing. 2.0 pmol F_DS-Cav1 antigen was added to half the wells of a 96-well plate for direct immobilization and blocking with bovine serum albumin. A series of 5-fold dilutions were prepared by appropriately adding buffer to the mouse immune serum or to the mouse immune serum after antigen adsorption and then added to the plate and incubated (at room temperature, 2 hours). Subsequently, the diluted serum was removed and coupled with a peroxidase-conjugated anti-mouse antigen antibody (DaKo, P0161), and then color developed using a standard peroxidase detection reagent, after which the intensity of color development was determined by measuring the absorbance on a standard plate reader. . The relationship between the rate of serum dilution and absorbance was calculated by piecewise linear interpolation between measurement points, and the reciprocal of the dilution rate at which absorbance was 0.2 was determined as the titer of the detected antibody against the F antigen, and the relative magnitude of the decrease in serum antibody titer after the adsorption procedure , if the titer of serum antibodies before the adsorption procedure was 100%, was determined as the adsorption rate (Fig. 14).

[0123]

Using the above procedure, it was shown that, compared with the F_DS-Cav1 antigen described in the literature, the FH_50, FH_81, FH_82 and FH_85 antigens have a low ability to induce post-F recognition antibody. That is, a result was obtained indicating that when using antigens FH_50, FH_81, FH_82, FH_85 as vaccine antigens, the risk of inducing an antibody that does not contribute to protection against viral infection is low.

[0124]

Example 6. Second comparison with predecessor

All F protein fragments of the FH_50, FH_81, FH_82 and FH_85 antigens obtained as described in Example 5 contain the L141C and L373C mutations. These mutations are mutations designed to introduce a disulfide bond as described in Example 1. However, an earlier patent (International Publication No. 2017/109629) assessed the introduction of a disulfide bond at a relatively close site (mutant ID: pXCS524.L142C, and N371C) and optimized antigens with additional mutations (mutant ID: pXCS881.L142C, N371C, S55C, L188C, T54H, V296I, D486S, E487Q, and D489S). Therefore, the beneficial antigens obtained as described above in the Examples were compared with the Pf524 or Pf881 antigen into which the pXCS524 mutation or the pXCS881 mutation was introduced. Descriptions of mutations in antigen precursor proteins obtained as described below are summarized in Table 6.

[0125]

Table 6

Antigen name Description of mutations Pf524 L142C, N371C FH_08 L141C, L373C Pf881 L142C, N317C, S55C, L188C, T54H, V296I, D486S, E487Q, D489S FH_82 L141C, L373C, R135N, R136N, T189V, S190V, K42R, V384T FH_85 L141C, L373C, R135N, R136N, T189V, S190V, E60M, K42R, V384T

[0126]

The antigens described in Example 1, Example 3 and Example 5, respectively, were used as antigens FH_08, FH_82 and FH85. Each of the polynucleotides encoding the Pf524 and Pf881 precursor proteins was inserted into the pcDNA3.4 vector (Thermo Fisher Scientific, Inc.) using standard genetic engineering techniques, and the insertion of the target DNA sequence was confirmed by base sequence analysis, resulting in whereby each of the mammalian cell expression vectors expressing antigen precursor proteins was constructed. The vector for introduction into the cell was purified using a standard plasmid vector purification method.

[0127]

Expression and purification of each of the antigens was carried out using the procedure described in Example 2.

The expression level of each antigen was assessed using the procedure described in Example 3.

[0128]

Antibody binding test

The ability of each antigen to bind to each antibody was assessed using the procedure described in Example 5. However, the binding signal value was set to the value obtained by subtracting the detection value if PBS (pH 7.4) was applied for 180 seconds from the detection value when applying each antigen solution for 180 seconds (Fig. 15-17).

[0129]

Using the above procedure, a result was obtained showing that for the FH_08 antigen, compared to the Pf524 antigen, which was introduced with the mutation described in the earlier patent, while the storage stability of the Φ epitope and trimer formation are excellent, the I epitope , which is known to induce an antibody with a rather low ability to neutralize viral infection, is practically undetectable. In addition, a result was obtained showing that similarly for the optimized antigens FH_82 and FH_85, compared to the Pf881 antigen, which was introduced with the mutation described in the earlier patent, while the storage stability of the Φ epitope and trimer formation were excellent , epitope I, which is known to induce an antibody with a rather low ability to neutralize viral infection, is practically undetectable. This suggests that the beneficial disulfide mutations described in the example above are mutations that preferentially induce a group of antibodies that are expected to have a stronger virus neutralizing effect compared to the disulfide mutations (L142C and N371C), described in an earlier patent.

[0130]

Confirmation of antibodies induced by murine immunity

Based on the above experiment, it was suggested that the FH_82 and FH_85 antigens induce immunity that does not neutralize the virus to a lesser extent than the Pf881 mutant, which was introduced with the mutation as described in the earlier patent. To study this effect in more detail, we analyzed antibodies induced by immunizing mice with the FH_82, FH_85 or Pf881 antigen.

[0131]

Analysis of the administered antibodies was carried out in accordance with the procedure described in Example 5, and the rate of production of antibody titer was measured using the ΔFP furin _{wt Fecto} antigen adsorption procedure (Fig. 18).

[0132]

In the above procedure, it was shown that, compared to the Pf881 antigen, which had been mutated as described in the earlier patent, the FH_82 and FH_85 antigens produced a lower level of induction of post-F recognition antibody. That is, this result suggests that when using FH_82 and FH_85 antigens as vaccine antigens, a group of antibodies are preferentially induced, which are considered to have a stronger virus neutralizing effect compared to Pf881.

[0133]

Example 7: Preparation of Antigen Particles Using Framework Particles

To further reduce the risk of inducing antibodies that do not contribute to protection against viral infection, it may be effective to reduce, through the effect of steric hindrance, the accessibility of antibodies or various immune cells to the antigenic site recognized by an antibody that recognizes post-F, such as an antibody that recognizes post-F. epitope I. Therefore, the possibility is considered that the effect of steric hindrance can be achieved by immobilizing antigens on appropriate framework particles, so that the recognition site of the post-F recognition antibody is on the inner side.

However, if the antigens are presented on suitable framework particles, if the density is low, that is, if the spaces between the antigens are equal to or greater than a certain length, or if flexible linkers of a certain length or greater are embedded between the antigens and the framework particles, then the described above the effect of steric hindrance cannot be achieved. Thus, GS linkers of varying lengths were evaluated using HBsAg VLPs, which are thought to be capable of providing relatively high-density antigen presentation.

[0134]

Preparation of antigen particles

Bionanocapsule-ZZ (Beacle Inc., BCL-DC-002), capable of binding to the Fc fusion protein, was used as framework particles.

The antigen precursor proteins used were the F_DS-Cav1-Fc antigen precursor protein (SEQ ID NO: 31), the FH_82-Fc antigen precursor protein (SEQ ID NO: 32), and the FH_85- antigen precursor protein (SEQ ID NO: 33). Fc, in each of which, the sequence (SEQ ID NO: 22) containing the thrombin recognition sequence, His tag, Strep tag II in the F_DS-Cav1, FH_82 and FH_85 precursor proteins described above was replaced by the Fc sequence (SEQ ID NO: 30). In addition, regarding the F_DS-Cav1-Fc antigen precursor protein and the FH_85-Fc precursor protein, these proteins in which the GS linker (GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 75), GGGGSGGGGS (SEQ ID NO: 19) or GGGGS (SEQ ID NO: 27)) inserted immediately before the Fc sequence were derived from the corresponding precursor proteins (SEQ ID NO: 34-39) using the following method.

[0135]

The polynucleotide encoding each of the above precursor proteins was inserted into the pcDNA3.4 vector (Thermo Fisher Scientific, Inc.) using standard genetic engineering techniques. The insertion of the target DNA sequence was confirmed by base sequence analysis, and mammalian cell expression vectors expressing antigen precursor proteins were each constructed. The vector for introduction into the cell was purified using a standard plasmid vector purification method.

[0136]

Expression of the precursor protein of each antigen was accomplished by expressing a secreted mammalian cell antigen using the Expi293 expression system (Thermo Fisher Scientific, Inc.). Each purified vector was introduced into an Expi293 cell using the Expi Fectamin 293 reagent, which is a gene transfer reagent from the same kit, and each encoded antigen was secreted and expressed by culturing for approximately 4-5 days, and the supernatant component was obtained by centrifugation and using a filter with a hole diameter of 0.22 µm.

Purification of each antigen was carried out using a standard affinity purification method on a protein A purification column. After purification, solvent exchange was performed with PBS (pH 7.4) using an ultrafiltration membrane.

Each of the prepared antigens was mixed with HBsAg VLPs at a ratio of approximately 135 molecules (approximately 45 molecules as trimer) per HBsAg VLP particle on average and immobilized onto the scaffold particles by keeping the mixture at room temperature for one hour. In this case, the almost complete absence of non-immobilized antigen was confirmed by performing electrophoresis in native PAGE with blue dye staining.

[0137]

The antigens used in the assay described in this Example and the particulate antigens obtained by immobilizing antigens on scaffold particles (which may also be referred to hereinafter as “particulate antigens”) are summarized in Table 7.

[0138]

[Table 7]

Antigen name Description F_DS-Cav1 Prepared as described in Example 5 F_DS-Cav1-Fc The sequence containing the thrombin recognition sequence, His tag, Strep tag II F_DS-Cav1 is replaced by the Fc sequence F_DS-Cav1-4GS-Fc GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 75) is inserted immediately before the Fc sequence F_DS-Cav1-Fc F_DS-Cav1-2GS-Fc GGGGSGGGGS (SEQ ID NO: 19) is inserted immediately before the Fc sequence F_DS-Cav1-Fc F_DS-Cav1-1GS-Fc GGGGS (SEQ ID NO: 27) is inserted immediately before the Fc sequence F_DS-Cav1-Fc F_DS-Cav1-VLP F_DS-Cav1-Fc immobilized on HBsAg VLPs F_DS-Cav1-4GS-VLP F_DS-Cav1-4GS-Fc immobilized on HBsAg VLPs F_DS-Cav1-2GS-VLP F_DS-Cav1-2GS-Fc immobilized on HBsAg VLP F_DS-Cav1-1GS-VLP F_DS-Cav1-1GS-Fc immobilized on HBsAg VLP FH_82 Prepared as described in Example 5 FH_82-Fc The sequence containing the thrombin recognition sequence, His tag, Strep tag II FH_82, is replaced by the Fc sequence FH_82-VLP FH_82-Fc immobilized on HBsAg VLP FH_85 Prepared as described in Example 5 FH_85-Fc The sequence containing the thrombin recognition sequence, His tag, Strep tag II FH_85, is replaced by the Fc sequence FH_85-4GS-Fc GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 75) is inserted immediately before the Fc sequence FH_85-Fc FH_85-2GS-Fc GGGGSGGGGS (SEQ ID NO: 19) is inserted immediately before the Fc sequence FH_85-Fc FH_85-1GS-Fc GGGGS (SEQ ID NO: 27) is replaced immediately before the Fc sequence FH_85-Fc FH_85-VLP FH_85-Fc immobilized on HBsAg VLP FH_85-4GS-VLP FH_85-4GS-Fc immobilized on HBsAg VLP FH_85-2GS-VLP FH_85-2GS-Fc immobilized on HBsAg VLP FH_85-1GS-VLP FH_85-1GS-Fc immobilized on HBsAg VLP

[0139]

Antibody binding test

The ability of F_DS-Cav1, F_DS-Cav1-VLP, FH_82, FH_82-VLP, FH_85 and FH_85-VLP, which are antigens or particulate antigens, to bind to antibodies was assessed using the procedure described in Example 6 (Fig. 19- 21).

After carrying out the above procedure, it was found that regardless of the method of pre-stabilization of the F antigen region (containing an amino acid substitution for pre-stabilization), if pre-F is immobilized on the framework particles through a domain attached to the C-terminus of its amino acid sequence, then trimer formation is improved, and at the same time, the ability of the antibody to bind to the Φ epitope is preserved. In addition, it has been shown that the magnitude at which epitope I, which is known to induce an antibody that has a rather low ability to neutralize viral infection, is detected is reduced. The above result suggests that if particulate antigens are used as vaccine antigens, a group of antibodies are preferentially induced which are believed to have a high ability to neutralize the virus.

[0140]

Linker Incorporation Assay

Ability F_DS-Cav1, F_DS-Cav1-4GS-VLP, F_DS-Cav1-2GS-VLP, F_DS-Cav1-1GS-VLP, F_DS-Cav1-VLP, FH_85, FH_85-4GS-VLP, FH_85-2GS-VLP, FH_85 -1GS-VLP and FH_85-VLP, which are antigens or particulate antigens, bind to antibodies, were assessed using the procedure described in Example 6 (Fig. 22-24).

After performing the above procedure, it was found that by incorporating any of the GS linkers, the effect of improving trimer formation was achieved while maintaining the ability of the antibody to bind to the Φ epitope and there was a decrease in the amount at which epitope I was detected. However, the effect of reducing the detection rate of epitope I was shown to be weakened if too long a GS linker is inserted between the framework particles and the antigen. In particular, no significant effect was observed at a length of approximately GGGGSGGGGS (SEQ ID NO: 19). However, for the longer sequence GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 75), a weakening effect of epitope I detection reduction was observed.

The above result indicates that if a flexible linker having a certain length or more is inserted between the framework particles and the antigen, the effect of reducing the accessibility of the post-F recognition antibody to the recognition site is weakened due to the effect of steric hindrance.

[0141]

Antigen density assessment

The theoretical particle diameter of the FH_85-VLP antigen particles was estimated using dynamic light scattering using a Zetasizer μV (Malvern) to be 89.28 nm (FIG. 25). Since there are an average of approximately 135 molecules (approximately 45 trimer molecules) per particle, the average molecular density per 1 nm ² theoretical surface area may be approximately 0.005 molecules (approximately 0.0018 trimer molecules). In this case, the surface area (S) of the sphere was calculated using the following formula using its radius (r): S=4πr ²

[0142]

That is, it is believed that the desired steric hindrance effect can be achieved if the density is at least about 0.005 antigen molecules or more per 1 nm ² theoretical surface area, as assessed using dynamic light scattering.

The same result was also obtained for DS-Cav1-VLP and FH_82-VLP (Fig. 25).

[0143]

Confirmation of antibodies induced by murine immunity

From the above experiment, it appears that pre-F antigen bound to HBsAg VLP scaffold particles has a suppressive effect in inducing immunity, which does not contribute to neutralization of the virus. To further examine this effect in more detail, antibodies induced by immunizing mice with F_DS-Cav1, F_DS-Cav1-VLP, FH_82, FH_82-VLP, FH_85, or FH_85-VLP antigens were analyzed.

Mouse immune sera were obtained by immunizing BALB/c mice (5-8 week old females) with particulate antigens. A thoroughly mixed solution of PBS or PBS with aluminum hydroxide (InvivoGen, vac-alu-250) in equal quantities was used as a composition of the solvent and each of the particulate antigens. The antigen was administered to mice intramuscularly in the hind thighs twice, approximately 3 weeks apart, and blood was collected approximately 3 weeks after the second administration. The resulting blood was incubated at room temperature and then centrifuged to collect the serum.

An ELISA assay in which the antigen of each conformation was immobilized was performed to confirm the extent of induction of antibody that binds to pre-F or post-F in serum.

In addition, to calculate the rate of induction of post-F recognition antibody, an antigen adsorption procedure was performed, and the rate of decrease in the level of antigen recognition antibody was measured by the antigen adsorption procedure. This method corresponds to the method described in Example 5.

In conclusion, due to the formation of particles, the effect of preferential induction of a group of antibodies that are considered to have a high ability to neutralize the virus is confirmed.

[0144]

[Example 8] Formation of antigen particles using a hydrophobic peptide chain

The possibility was considered that the effect of reducing the accessibility of antibodies or various immune cells to the site of the antigen recognized by the post-F antibody, as shown in Example 7, due to the effect of steric hindrance, could also be achieved using a method that does not use frame particles. Therefore, the present Example describes the evaluation of particle formation by hydrophobic aggregation using a hydrophobic peptide chain (a polypeptide chain containing a hydrophobic amino acid).

[0145]

Hydrophobic Peptide Chain Evaluation

The hydrophobic peptide chain used should be capable of hydrophobic aggregation in the vaccine preparation or in vivo conditions, but it is difficult to apply in practice for a sequence that significantly reduces the efficiency of secretory expression of the antigen by cells. In general, it is known that when a polypeptide chain containing a large number of hydrophobic amino acids is fused with a protein, the efficiency of protein production in cells decreases. Therefore, suitable hydrophobic polypeptide chains were investigated.

The antigens were derived from precursor proteins in which the sequence (SEQ ID NO: 22) containing the thrombin recognition sequence, His tag and Strep tag II in the F_DS-Cav1 antigen precursor protein was replaced by sequences (SEQ ID NO: 44-47) that contain various hydrophobic peptide chains (SEQ ID NO: 40-43) and a FLAG tag, which is a purification tag. The studied sequences of hydrophobic peptides are systematized in Table 8-1.

[0146]

The polynucleotide encoding each of the above precursor proteins was inserted into the pcDNA3.4 vector (Thermo Fisher Scientific, Inc.) using standard genetic engineering techniques. The insertion of the target DNA sequence was confirmed by base sequence analysis, and mammalian cell expression vectors expressing antigen precursor proteins were each constructed. The vector for introduction into the cell was purified using a standard plasmid vector purification method.

[0147]

Expression of each of the antigens was carried out in accordance with the procedure described in Example 7.

Purification of each antigen was performed by affinity purification using an affinity gel containing anti-FLAG M2 antibody (Sigma, A2220). The FLAG peptide was used for elution in affinity purification. However, the FLAG peptide in each antigen sample was removed using an ultrafiltration membrane. PBS (pH 7.4) was used as a solvent.

The ability to express the secreted antigen was determined by performing total SDS-PAGE after purification and confirming that the target protein could be isolated and purified with a high degree of purity. In addition, the possibility of particle formation was confirmed by performing conventional native PAGE with the addition of blue dye after purification.

[0148]

Table 8-1

Amino acid sequence of the hydrophobic peptide chain used CC02 I A L A L E TO I A L A L E TO I A L A L E TO (SEQ ID NO.: 40) CC03 I TO L E L E TO I TO L E L E TO I TO L E L E TO (SEQ ID NO.: 41) CC07 L H L H I E TO L H L H I E TO L H L H I E TO (SEQ ID NO.: 42) CC08 L TO L E I H H L TO L E I H H L TO L E I H H (SEQ ID NO.: 43) Note: I and L are highly hydrophobic amino acids. K and E are hydrophilic amino acids.

[0149]

The secreted antigen expression and particle formation capabilities of F_DS-Cav1-CC02 and F_DS-Cav1-CC03 were examined and it was found that F_DS-Cav1-CC03 is secreted and expressed but does not form particles, and F_DS-Cav1-CC02 is not sufficiently secreted and expressed (Fig. 26). Therefore, it is believed that the optimal hydrophobicity of the hydrophobic peptide chain used lies between the hydrophobicities of the two antigens.

The difference between the hydrophobic peptide chains of F_DS-Cav1-CC02 and F_DS-Cav1-CC03 is that alanine, which is a non-hydrophilic amino acid, is replaced by lysine or glutamic acid, which is a hydrophilic amino acid. Therefore, it was first decided to replace the F_DS-Cav1-CC02 alanine with histidine, which is moderately hydrophilic and has a pK corresponding to approximately pH 6. Since it is known that the pH of the solvent in the preparation of a pharmaceutical drug is close to neutral, and the pH in the secretory pathway of cells is more low, it was expected that the histidine-containing peptide chain would have a relatively hydrophilic property during expression of the secreted antigen and a relatively hydrophobic property during the preparation of the pharmaceutical composition.

In addition, given that the engineered hydrophobic peptide chain forms a supercoiled structure, the 21 amino acid hydrophobic peptide chain fragments connected directly in the presence of the foldon domain are expected to be defgabcdefgabcdefgabc in that order. If leucine and isoleucine are located at positions (a) and (d), which interact hydrophobically in the trimer, according to a previous study (Joel P. Schneider et al., 1998, Folding & Design, 3:R29), which evaluated the amino acid sequences of natural proteins, since isoleucine at position (a) and leucine at position (d) are more common, it is considered that a suitable replacement is the replacement of leucine and isoleucine at position (a) and at position (d) F_DS-Cav1-CC02.

Based on the above, F_DS-Cav1-CC07 was newly constructed, and the secreted antigen expression capability and particle formation capability were investigated, and both secreted antigen expression and particle formation were achieved (FIG. 27). Since the expression of secreted antigen is difficult for F_DS-Cav1-CC08, in which the positions of histidine, lysine and glutamic acid are replaced, it has also been suggested that the problem lies not only in the amino acid composition, but also in achieving a three-dimensional rearrangement.

[Chemical formula 1]

(taken from Folding & Design 1998, 3:R29)

[0150]

(Preparation of particulate antigen)

The antigens were derived from precursor proteins (SEQ ID NO: 48 and 49), in which the sequence (SEQ ID NO: 22) containing the thrombin recognition sequence, His tag and Strep tag II in the precursor proteins of the FH_82 and FH_85 antigens, has been replaced by a sequence that contains an optimal hydrophobic peptide chain (SEQ ID NO: 42) and a FLAG tag, which is a purification tag.

[0151]

The polynucleotide encoding the above precursor proteins was inserted into the pcDNA3.4 vector (Thermo Fisher Scientific, Inc.) using standard genetic engineering techniques. The insertion of the target DNA sequence was confirmed by base sequence analysis, and mammalian cell expression vectors expressing the precursor proteins were each constructed. The vector for introduction into the cell was purified using a standard plasmid vector purification method.

[0152]

Expression of each antigen was carried out according to the procedure described in the hydrophobic peptide chain assay.

Purification of each antigen was performed according to the procedure described in the Hydrophobic Peptide Chain Analysis.

[0153]

The antigens (which may also be referred to as “particulate antigens”) used in the assay described in this Example are summarized in Table 8-2.

[0154]

Table 8-2

Antigen name Description F_DS-Cav1 Prepared as described in Example 5 F_DS-Cav1-CC07 The sequence containing the thrombin recognition sequence, His tag, Strep II tag F_DS-Cav1 is replaced by a sequence containing the hydrophobic peptide chain "LHLHIEKLHLHIEKLHLHIEK" (SEQ ID NO: 42) and the FLAG tag. FH_82 Prepared as described in Example 5 FH_82-CC07 The sequence containing the thrombin recognition sequence, His tag, Strep tag II FH_82, is replaced by a sequence containing the hydrophobic peptide chain "LHLHIEKLHLHIEKLHLHIEK" (SEQ ID NO: 42) and a FLAG tag. FH_85 Prepared as described in Example 5 FH_85-CC07 The sequence containing the thrombin recognition sequence, His tag, Strep II tag FH_85 is replaced by a sequence containing the hydrophobic peptide chain "LHLHIEKLHLHIEKLHLHIEK" (SEQ ID NO: 42) and the FLAG tag.

[0155]

Antibody binding assay

The ability of the resulting particulate antigens to bind to antibodies was assessed according to the procedure described in Example 6 (Fig. 28-30).

After performing the above procedure, it was found that regardless of the method of pre-stabilization of the F antigen region (containing an amino acid substitution for pre-stabilization), a particulate antigen aggregated with a hydrophobic peptide chain having a pre-F added to the C-terminus of its amino acid sequence improves formation of a trimer while maintaining the ability of the antibody to bind to the Φ epitope. In addition, a result was obtained showing that the detection of epitope I, which is known to induce an antibody having a rather low ability to neutralize viral infection, is reduced. The above results suggest that when using the antigen as a vaccine antigen, the effect of preferentially inducing a group of antibodies believed to have a high ability to neutralize the virus is improved by particle formation.

[0156]

Antigen density assessment

The theoretical diameters of FH_85 antigen and particulate antigen FH_85-CC07 were estimated using dynamic light scattering using a DynaPro Plate Reader III (Wyatt) to be 13.8 nm and 60.0 nm (Figure 31). Since the ratio of the molecular weights of the two particles was proportional to the ratio of their theoretical diameters to the 2nd and 3rd powers for conventional proteins, the molecular weight of FH_85-CC07 is estimated to be approximately 20-80 times that of FH_85. That is, FH85-CC07 is believed to be approximately 60-240 molecules per particle (approximately 20-80 molecules as a trimer), and the average molecular density per 1 ^nm2 theoretical surface area may be approximately 0.005-0.022 molecules (approximately 0. 0017-0.0073 molecules in the form of a trimer).

The same results were obtained for FH_82-CC07 (Fig. 31).

Thus, it was found that the particulate antigen produced using the hydrophobic peptide chain has an antigen density equivalent to or greater than the density of the particulate antigen produced using the framework particles presented in Example 6.

[0157]

Confirmation of antibodies induced by murine immunity

Using the above procedure, it has been suggested that the formation of antigen particles to which a hydrophobic peptide chain is bound has a suppressive effect in inducing immunity that does not contribute to neutralization of the virus. To study this effect in more detail, we analyzed antibodies induced by immunizing mice with antigen or particulate antigen F_DS-Cav1F_DS-Cav1-CC07, FH_82-CC07, or FH_85-CC07.

[0158]

Mouse immune sera were obtained by immunizing BALB/cAnNCrlCrlj (Charles River Laboratories Japan) (7-week-old females) with antigens and particulate antigens. Antigen or particulate antigen F_DS-Cav1, F_DS-Cav1-CC07, FH_82-CC07 or FH_85-CC07 (antigen concentration: 0.40 mg/ml), dissolved in PBS (pH 7.4), was administered intramuscularly in an amount of 50 µl into the posterior thigh of mice on the 0th day of immunization and on the 21st day of immunization (5 mice per group). Blood was collected from the posterior vena cava on day 42 of immunization, and the resulting blood was incubated (at room temperature, approximately 2 hours, under stationary conditions), and then the serum was collected by centrifugation and inactivated (at 55°C, 30 minutes) for preparation mouse immune serum.

[0159]

To confirm the ratio at which antibodies are induced that bind to postfusion conformations, using the method described in Example 6, each of the mouse immune sera was subjected to antigen adsorption using magnetic beads to which ΔFP furin was attached_wtF_ecto, or magnetic beads that have only been subjected to a blocking procedure without binding to any antigen. In addition, for each of the sera, after the adsorption procedure, an ELISA assay was performed in which F_DS-Cav1 was immobilized using the method described in Example 6, and the adsorption rate was calculated. However, the adsorption rate was set equal to the relative value obtained by subtracting the antibody titer in the serum after the adsorption procedure using the ΔFP furin antigen_wtF_ecto from the antibody titer in the serum after the adsorption procedure but without the antigen, if the serum antibody titer after the adsorption procedure without the antigen was taken as 100% (Fig. 32).

[0160]

After performing the above procedure, it was shown that the post-F recognition antibody, which was approximately half induced by the F_DS-Cav1 antigen before particle formation, was almost not induced by the F_DS-Cav1-CC07 antigen, which forms particles through the hydrophobic peptide chain. Moreover, similarly, the post-F recognition antibody was also hardly induced by the FH_82-CC07 and FH_85-CC07 antigens. Based on the above, it is assumed that by forming particles, the effect of preferentially inducing a group of antibodies that are considered to have a high ability to neutralize the virus is improved.

[0161]

[Example 9] Formation of Antigen Particles Using a Self-Associating Protein Domain

The possibility was considered that the effect of reducing the accessibility of antibodies or various immune cells to the antigenic site recognized by the post-F recognition antibody, as shown in Examples 7 and 8, due to the effect of steric hindrance, could also be realized by fusing the self-associating protein domain with C -end side of the antigen. In particular, due to the regular arrangement of antigens using a self-associating protein domain, the possibility of achieving a higher effect of steric hindrance than the formation of particles by hydrophobic aggregation, as shown in Example 8, was considered.

Thus, in this Example, particle formation was examined using the hepatitis B virus core antigen (HBcAg), which has the ability to self-associate.

However, in cases where the sequence (SEQ ID NO: 22) containing the thrombin recognition sequence, His tag and Strep tag II in the F_DS-Cav1 antigen was replaced with a peptide sequence (SEQ ID NO: 51), which contains a GS linker (GGGGSGGGGSGGGGSGGGGS) (SEQ ID NO: 75), a self-associating HBcAg domain (SEQ ID NO: 50) and a His tag, then, despite the observed expression, self-association was not detected (the result of this analysis was confirmed using electrophoresis in the native PAGE with blue dye). The N-terminus of the self-associating domain of HBcAg is present near the binding surface if HbcAg undergoes self-association, and the possibility has been considered that self-association may be impeded by the fusion of a large protein to the N-terminus.

Thus, to position the RSV F antigen fragment at a position away from the binding surface during self-association, if HBcAg undergoes such self-association, it must be divided into a C-terminal side and an N-terminal side, but not into the outward-protruding side of the particle ( fragment between the helices present in domain α3 and domain α4). Additionally, to reduce the risk of unexpected disulfide bond formation, two point mutations (C48S and C107A) were introduced that are not expected to affect self-association. Specifically, antigens (which may also be referred to as “particulate antigens”) were prepared from the precursor protein (SEQ ID NO: 55) F_DS-Cav1-HBcCN and the precursor protein (SEQ ID NO: 56) FH_85-HBcCN, in each of which the sequence (SEQ ID NO: 22) containing the thrombin recognition sequence, His tag and Strep tag II in the precursor proteins of the F_DS-Cav1 and FH_85 antigens was replaced by a sequence containing the GS linker (GGGGSGGGGSGGGGSGGGGS) (SEQ ID NO : 75), the C-terminal side of the HBcAg self-associating domain (SEQ ID NO: 52) and the sequence (SEQ ID NO: 54) in which the GS linker (GGGGSGGGGSGGGGSGGGGS) (SEQ ID NO: 20), the N-terminal side of the HBcAg self-associating domain (SEQ ID NO: 53) and the FLAG tag are associated in the order shown.

F_DS-Cav1-HBcCN was expressed and purified using the procedure described in Example 8, and the ability to express secreted antigen and the ability to form particles was examined, resulting in both secreted antigen expression and particle formation (FIG. 33). However, since the relative expression level was low, an improvement in the expression method would be desirable. Therefore, for the polynucleotide encoding the precursor DS-Cav1-HBcCN or FH_85-HBcCN, a stable expression CHO line was generated using the GS Xceed gene expression system (Lonza). The resulting cell line was cultured, and after secretion and expression of each antigen, a component of the culture supernatant was obtained.

Purification of each particulate antigen was carried out using the procedure described in Example 8.

To confirm the ability of each particulate antigen to bind to each antibody, an ELISA assay was performed in which palivizumab (Synagis intramuscular injection, AbbVie, Inc.), antibody 131-2A, and antibody D25 were immobilized. Carbonate-bicarbonate buffer (Sigma-Aldrich, C3041-50CAP) was used as antibody binding buffer, and TBS (TaKaRa, T9142) was used for sample dilution and plate washing. 10 μg/ml palivizumab or antibody 131-2A or antibody D25 was added to half the wells of a 96-well plate for direct immobilization and blocking with bovine serum albumin. A series of 10-fold dilutions were prepared by appropriately adding buffer to each antigen and added to the plate for incubation (at room temperature, 2 hours, under stationary conditions). Thereafter, the added antigen solution was removed and coupled with peroxidase-conjugated anti-FLAG antibody M2 (Sigma-Aldrich, A8592), and then color developed using a standard peroxidase detection reagent, after which the intensity of color development was determined by measuring the absorbance on a standard tablet reader. The relationship between the rate of antigen dilution and absorbance was calculated by piecewise linear interpolation between measurement points, and the reciprocal of the dilution rate at which absorbance equals 0.2 was used as the binding strength of the added antigen to the immobilized antibody. The value obtained by dividing the binding strength of antibody D25 by the binding strength of palivizumab was defined as the epitope detection signal Φ, and the value obtained by dividing the binding strength of antibody 131-2A by the binding strength of palivizumab was defined as the epitope detection signal I ( Fig. 34 and 35).

Using the above procedure, a result was obtained showing that, compared with the formation of particles through a hydrophobic peptide chain, the particulate antigen aggregated by the action of a self-associating protein domain with a pre-F added to the C-terminus of the amino acid sequence also markedly improves the ability antibodies bind to the Φ epitope and reduces the detection rate of the I epitope, which is known to induce an antibody that has a rather low ability to neutralize viral infection. The above result indicates that the effect of preferentially inducing a group of antibodies believed to be highly active in neutralizing the virus is improved by particle formation.

--->

LIST OF SEQUENCES

<110> Mitsubishi Tanabe Pharma Corporation

<120> Mutant F protein of RSV and its application

<130> 19084-201016

<150>US62/952673

<151> 2019-12-23

<160> 75

<170> Patent In version 3.5

<210> 1

<211> 574

<212>PRT

<213> Respiratory syncytial virus

<220>

<221> MISC_FEATURE

<222> (1)..(25)

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<220>

<221> MISC_FEATURE

<222> (26)..(109)

<223> F2

<220>

<221> MISC_FEATURE

<222> (110)..(136)

<223> pep27

<220>

<221> MISC_FEATURE

<222> (137)..(513)

<223> extracellular F1

<220>

<221> MISC_FEATURE

<222> (514)..(574)

<223> transmembrane and intracellular F1

<400> 1

Met Glu Leu Leu Ile Leu Lys Ala Asn Ala Ile Thr Thr Ile Leu Thr

1 5 10 15

Ala Val Thr Phe Cys Phe Ala Ser Gly Gln Asn Ile Thr Glu Glu Phe

20 25 30

Tyr Gln Ser Thr Cys Ser Ala Val Ser Lys Gly Tyr Leu Ser Ala Leu

35 40 45

Arg Thr Gly Trp Tyr Thr Ser Val Ile Thr Ile Glu Leu Ser Asn Ile

50 55 60

Lys Glu Asn Lys Cys Asn Gly Thr Asp Ala Lys Val Lys Leu Ile Lys

65 70 75 80

Gln Glu Leu Asp Lys Tyr Lys Asn Ala Val Thr Glu Leu Gln Leu Leu

85 90 95

Met Gln Ser Thr Pro Pro Thr Asn Asn Arg Ala Arg Arg Glu Leu Pro

100 105 110

Arg Phe Met Asn Tyr Thr Leu Asn Asn Ala Lys Lys Thr Asn Val Thr

115 120 125

Leu Ser Lys Lys Arg Lys Arg Arg Phe Leu Gly Phe Leu Leu Gly Val

130 135 140

Gly Ser Ala Ile Ala Ser Gly Val Ala Val Ser Lys Val Leu His Leu

145 150 155 160

Glu Gly Glu Val Asn Lys Ile Lys Ser Ala Leu Leu Ser Thr Asn Lys

165 170 175

Ala Val Val Ser Leu Ser Asn Gly Val Ser Val Leu Thr Ser Lys Val

180 185 190

Leu Asp Leu Lys Asn Tyr Ile Asp Lys Gln Leu Leu Pro Ile Val Asn

195 200 205

Lys Gln Ser Cys Ser Ile Ser Asn Ile Glu Thr Val Ile Glu Phe Gln

210 215 220

Gln Lys Asn Asn Arg Leu Leu Glu Ile Thr Arg Glu Phe Ser Val Asn

225 230 235 240

Ala Gly Val Thr Thr Pro Val Ser Thr Tyr Met Leu Thr Asn Ser Glu

245 250 255

Leu Leu Ser Leu Ile Asn Asp Met Pro Ile Thr Asn Asp Gln Lys Lys

260 265 270

Leu Met Ser Asn Asn Val Gln Ile Val Arg Gln Gln Ser Tyr Ser Ile

275 280 285

Met Ser Ile Ile Lys Glu Glu Val Leu Ala Tyr Val Val Gln Leu Pro

290 295 300

Leu Tyr Gly Val Ile Asp Thr Pro Cys Trp Lys Leu His Thr Ser Pro

305 310 315 320

Leu Cys Thr Thr Asn Thr Lys Glu Gly Ser Asn Ile Cys Leu Thr Arg

325 330 335

Thr Asp Arg Gly Trp Tyr Cys Asp Asn Ala Gly Ser Val Ser Phe Phe

340 345 350

Pro Gln Ala Glu Thr Cys Lys Val Gln Ser Asn Arg Val Phe Cys Asp

355 360 365

Thr Met Asn Ser Leu Thr Leu Pro Ser Glu Ile Asn Leu Cys Asn Val

370 375 380

Asp Ile Phe Asn Pro Lys Tyr Asp Cys Lys Ile Met Thr Ser Lys Thr

385 390 395 400

Asp Val Ser Ser Ser Val Ile Thr Ser Leu Gly Ala Ile Val Ser Cys

405 410 415

Tyr Gly Lys Thr Lys Cys Thr Ala Ser Asn Lys Asn Arg Gly Ile Ile

420 425 430

Lys Thr Phe Ser Asn Gly Cys Asp Tyr Val Ser Asn Lys Gly Met Asp

435 440 445

Thr Val Ser Val Gly Asn Thr Leu Tyr Tyr Val Asn Lys Gln Glu Gly

450 455 460

Lys Ser Leu Tyr Val Lys Gly Glu Pro Ile Ile Asn Phe Tyr Asp Pro

465 470 475 480

Leu Val Phe Pro Ser Asp Glu Phe Asp Ala Ser Ile Ser Gln Val Asn

485 490 495

Glu Lys Ile Asn Gln Ser Leu Ala Phe Ile Arg Lys Ser Asp Glu Leu

500 505 510

Leu His Asn Val Asn Ala Gly Lys Ser Thr Thr Asn Ile Met Ile Thr

515 520 525

Thr Ile Ile Ile Val Ile Ile Val Ile Leu Leu Ser Leu Ile Ala Val

530 535 540

Gly Leu Leu Leu Tyr Cys Lys Ala Arg Ser Thr Pro Val Thr Leu Ser

545 550 555 560

Lys Asp Gln Leu Ser Gly Ile Asn Asn Ile Ala Phe Ser Asn

565 570

<210> 2

<211> 461

<212>PRT

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<220>

<221> MISC_FEATURE

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<220>

<221> MISC_FEATURE

<222> (85)..(461)

<223> extracellular F1

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Gln Asn Ile Thr Glu Glu Phe Tyr Gln Ser Thr Cys Ser Ala Val Ser

1 5 10 15

Lys Gly Tyr Leu Ser Ala Leu Arg Thr Gly Trp Tyr Thr Ser Val Ile

20 25 30

Thr Ile Glu Leu Ser Asn Ile Lys Glu Asn Lys Cys Asn Gly Thr Asp

35 40 45

Ala Lys Val Lys Leu Ile Lys Gln Glu Leu Asp Lys Tyr Lys Asn Ala

50 55 60

Val Thr Glu Leu Gln Leu Leu Met Gln Ser Thr Pro Pro Thr Asn Asn

65 70 75 80

Arg Ala Arg Arg Phe Leu Gly Phe Leu Leu Gly Val Gly Ser Ala Ile

85 90 95

Ala Ser Gly Val Ala Val Ser Lys Val Leu His Leu Glu Gly Glu Val

100 105 110

Asn Lys Ile Lys Ser Ala Leu Leu Ser Thr Asn Lys Ala Val Val Ser

115 120 125

Leu Ser Asn Gly Val Ser Val Leu Thr Ser Lys Val Leu Asp Leu Lys

130 135 140

Asn Tyr Ile Asp Lys Gln Leu Leu Pro Ile Val Asn Lys Gln Ser Cys

145 150 155 160

Ser Ile Ser Asn Ile Glu Thr Val Ile Glu Phe Gln Gln Lys Asn Asn

165 170 175

Arg Leu Leu Glu Ile Thr Arg Glu Phe Ser Val Asn Ala Gly Val Thr

180 185 190

Thr Pro Val Ser Thr Tyr Met Leu Thr Asn Ser Glu Leu Leu Ser Leu

195 200 205

Ile Asn Asp Met Pro Ile Thr Asn Asp Gln Lys Lys Leu Met Ser Asn

210 215 220

Asn Val Gln Ile Val Arg Gln Gln Ser Tyr Ser Ile Met Ser Ile Ile

225 230 235 240

Lys Glu Glu Val Leu Ala Tyr Val Val Gln Leu Pro Leu Tyr Gly Val

245 250 255

Ile Asp Thr Pro Cys Trp Lys Leu His Thr Ser Pro Leu Cys Thr Thr

260 265 270

Asn Thr Lys Glu Gly Ser Asn Ile Cys Leu Thr Arg Thr Asp Arg Gly

275 280 285

Trp Tyr Cys Asp Asn Ala Gly Ser Val Ser Phe Phe Pro Gln Ala Glu

290 295 300

Thr Cys Lys Val Gln Ser Asn Arg Val Phe Cys Asp Thr Met Asn Ser

305 310 315 320

Leu Thr Leu Pro Ser Glu Ile Asn Leu Cys Asn Val Asp Ile Phe Asn

325 330 335

Pro Lys Tyr Asp Cys Lys Ile Met Thr Ser Lys Thr Asp Val Ser Ser

340 345 350

Ser Val Ile Thr Ser Leu Gly Ala Ile Val Ser Cys Tyr Gly Lys Thr

355 360 365

Lys Cys Thr Ala Ser Asn Lys Asn Arg Gly Ile Ile Lys Thr Phe Ser

370 375 380

Asn Gly Cys Asp Tyr Val Ser Asn Lys Gly Met Asp Thr Val Ser Val

385 390 395 400

Gly Asn Thr Leu Tyr Tyr Val Asn Lys Gln Glu Gly Lys Ser Leu Tyr

405 410 415

Val Lys Gly Glu Pro Ile Ile Asn Phe Tyr Asp Pro Leu Val Phe Pro

420 425 430

Ser Asp Glu Phe Asp Ala Ser Ile Ser Gln Val Asn Glu Lys Ile Asn

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Gln Ser Leu Ala Phe Ile Arg Lys Ser Asp Glu Leu Leu

450 455 460

<210> 3

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<221> MISC_FEATURE

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<221> MISC_FEATURE

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Gln Asn Ile Thr Glu Glu Phe Tyr Gln Ser Thr Cys Ser Ala Val Ser

1 5 10 15

Lys Gly Tyr Leu Ser Ala Leu Arg Thr Gly Trp Tyr Thr Ser Val Ile

20 25 30

Thr Ile Glu Leu Ser Asn Ile Lys Glu Asn Lys Cys Asn Gly Thr Asp

35 40 45

Ala Lys Val Lys Leu Ile Lys Gln Glu Leu Asp Lys Tyr Lys Asn Ala

50 55 60

Val Thr Glu Leu Gln Leu Leu Met Gln Ser Thr Pro Pro Thr Asn Asn

65 70 75 80

Arg Ala Arg Arg Glu Leu Pro Arg Phe Met Asn Tyr Thr Leu Asn Asn

85 90 95

Ala Lys Lys Thr Asn Val Thr Leu Ser Lys Lys Arg Lys Arg Arg Phe

100 105 110

Leu Gly Phe Leu Leu Gly Val Gly Ser Ala Ile Ala Ser Gly Val Ala

115 120 125

Val Ser Lys Val Leu His Leu Glu Gly Glu Val Asn Lys Ile Lys Ser

130 135 140

Ala Leu Leu Ser Thr Asn Lys Ala Val Val Ser Leu Ser Asn Gly Val

145 150 155 160

Ser Val Leu Thr Ser Lys Val Leu Asp Leu Lys Asn Tyr Ile Asp Lys

165 170 175

Gln Leu Leu Pro Ile Val Asn Lys Gln Ser Cys Ser Ile Ser Asn Ile

180 185 190

Glu Thr Val Ile Glu Phe Gln Gln Lys Asn Asn Arg Leu Leu Glu Ile

195 200 205

Thr Arg Glu Phe Ser Val Asn Ala Gly Val Thr Thr Pro Val Ser Thr

210 215 220

Tyr Met Leu Thr Asn Ser Glu Leu Leu Ser Leu Ile Asn Asp Met Pro

225 230 235 240

Ile Thr Asn Asp Gln Lys Lys Leu Met Ser Asn Asn Val Gln Ile Val

245 250 255

Arg Gln Gln Ser Tyr Ser Ile Met Ser Ile Ile Lys Glu Glu Val Leu

260 265 270

Ala Tyr Val Val Gln Leu Pro Leu Tyr Gly Val Ile Asp Thr Pro Cys

275 280 285

Trp Lys Leu His Thr Ser Pro Leu Cys Thr Thr Asn Thr Lys Glu Gly

290 295 300

Ser Asn Ile Cys Leu Thr Arg Thr Asp Arg Gly Trp Tyr Cys Asp Asn

305 310 315 320

Ala Gly Ser Val Ser Phe Phe Pro Gln Ala Glu Thr Cys Lys Val Gln

325 330 335

Ser Asn Arg Val Phe Cys Asp Thr Met Asn Ser Leu Thr Leu Pro Ser

340 345 350

Glu Ile Asn Leu Cys Asn Val Asp Ile Phe Asn Pro Lys Tyr Asp Cys

355 360 365

Lys Ile Met Thr Ser Lys Thr Asp Val Ser Ser Ser Val Ile Thr Ser

370 375 380

Leu Gly Ala Ile Val Ser Cys Tyr Gly Lys Thr Lys Cys Thr Ala Ser

385 390 395 400

Asn Lys Asn Arg Gly Ile Ile Lys Thr Phe Ser Asn Gly Cys Asp Tyr

405 410 415

Val Ser Asn Lys Gly Met Asp Thr Val Ser Val Gly Asn Thr Leu Tyr

420 425 430

Tyr Val Asn Lys Gln Glu Gly Lys Ser Leu Tyr Val Lys Gly Glu Pro

435 440 445

Ile Ile Asn Phe Tyr Asp Pro Leu Val Phe Pro Ser Asp Glu Phe Asp

450 455 460

Ala Ser Ile Ser Gln Val Asn Glu Lys Ile Asn Gln Ser Leu Ala Phe

465 470 475 480

Ile Arg Lys Ser Asp Glu Leu Leu

485

<210> 4

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<212>PRT

<213> Artificial sequence

<220>

<223> native F

<220>

<221> MISC_FEATURE

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<220>

<221> MISC_FEATURE

<222> (26)..(109)

<223> F2

<220>

<221> MISC_FEATURE

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<220>

<221> MISC_FEATURE

<222> (137)..(513)

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<220>

<221> MISC_FEATURE

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<220>

<221> MISC_FEATURE

<222> (543)..(562)

<223> label

<400> 4

Met Glu Leu Leu Ile Leu Lys Ala Asn Ala Ile Thr Thr Ile Leu Thr

1 5 10 15

Ala Val Thr Phe Cys Phe Ala Ser Gly Gln Asn Ile Thr Glu Glu Phe

20 25 30

Tyr Gln Ser Thr Cys Ser Ala Val Ser Lys Gly Tyr Leu Ser Ala Leu

35 40 45

Arg Thr Gly Trp Tyr Thr Ser Val Ile Thr Ile Glu Leu Ser Asn Ile

50 55 60

Lys Glu Asn Lys Cys Asn Gly Thr Asp Ala Lys Val Lys Leu Ile Lys

65 70 75 80

Gln Glu Leu Asp Lys Tyr Lys Asn Ala Val Thr Glu Leu Gln Leu Leu

85 90 95

Met Gln Ser Thr Pro Ala Thr Asn Asn Arg Ala Arg Arg Glu Leu Pro

100 105 110

Arg Phe Met Asn Tyr Thr Leu Asn Asn Ala Lys Lys Thr Asn Val Thr

115 120 125

Leu Ser Lys Lys Arg Lys Arg Arg Phe Leu Gly Phe Leu Leu Gly Val

130 135 140

Gly Ser Ala Ile Ala Ser Gly Val Ala Val Ser Lys Val Leu His Leu

145 150 155 160

Glu Gly Glu Val Asn Lys Ile Lys Ser Ala Leu Leu Ser Thr Asn Lys

165 170 175

Ala Val Val Ser Leu Ser Asn Gly Val Ser Val Leu Thr Ser Lys Val

180 185 190

Leu Asp Leu Lys Asn Tyr Ile Asp Lys Gln Leu Leu Pro Ile Val Asn

195 200 205

Lys Gln Ser Cys Ser Ile Ser Asn Ile Glu Thr Val Ile Glu Phe Gln

210 215 220

Gln Lys Asn Asn Arg Leu Leu Glu Ile Thr Arg Glu Phe Ser Val Asn

225 230 235 240

Ala Gly Val Thr Thr Pro Val Ser Thr Tyr Met Leu Thr Asn Ser Glu

245 250 255

Leu Leu Ser Leu Ile Asn Asp Met Pro Ile Thr Asn Asp Gln Lys Lys

260 265 270

Leu Met Ser Asn Asn Val Gln Ile Val Arg Gln Gln Ser Tyr Ser Ile

275 280 285

Met Ser Ile Ile Lys Glu Glu Val Leu Ala Tyr Val Val Gln Leu Pro

290 295 300

Leu Tyr Gly Val Ile Asp Thr Pro Cys Trp Lys Leu His Thr Ser Pro

305 310 315 320

Leu Cys Thr Thr Asn Thr Lys Glu Gly Ser Asn Ile Cys Leu Thr Arg

325 330 335

Thr Asp Arg Gly Trp Tyr Cys Asp Asn Ala Gly Ser Val Ser Phe Phe

340 345 350

Pro Gln Ala Glu Thr Cys Lys Val Gln Ser Asn Arg Val Phe Cys Asp

355 360 365

Thr Met Asn Ser Leu Thr Leu Pro Ser Glu Val Asn Leu Cys Asn Val

370 375 380

Asp Ile Phe Asn Pro Lys Tyr Asp Cys Lys Ile Met Thr Ser Lys Thr

385 390 395 400

Asp Val Ser Ser Ser Val Ile Thr Ser Leu Gly Ala Ile Val Ser Cys

405 410 415

Tyr Gly Lys Thr Lys Cys Thr Ala Ser Asn Lys Asn Arg Gly Ile Ile

420 425 430

Lys Thr Phe Ser Asn Gly Cys Asp Tyr Val Ser Asn Lys Gly Val Asp

435 440 445

Thr Val Ser Val Gly Asn Thr Leu Tyr Tyr Val Asn Lys Gln Glu Gly

450 455 460

Lys Ser Leu Tyr Val Lys Gly Glu Pro Ile Ile Asn Phe Tyr Asp Pro

465 470 475 480

Leu Val Phe Pro Ser Asp Glu Phe Asp Ala Ser Ile Ser Gln Val Asn

485 490 495

Glu Lys Ile Asn Gln Ser Leu Ala Phe Ile Arg Lys Ser Asp Glu Leu

500 505 510

Leu Gly Ser Gly Tyr Ile Pro Glu Ala Pro Arg Asp Gly Gln Ala Tyr

515 520 525

Val Arg Lys Asp Gly Glu Trp Val Leu Leu Ser Thr Phe Leu Leu Val

530 535 540

Pro Arg Gly Ser His His His His His Trp Ser His Pro Gln Phe

545 550 555 560

Glu Lys

<210> 5

<211> 1686

<212> DNA

<213> Artificial sequence

<220>

<223> native F

<400> 5

atggaactcc tcattcttaa ggctaacgcc ataaccacga ttctgactgc tgtgaccttc 60

tgctttgcta gcgggcaaaa cattaccgaa gagttctatc aatccacttg tagcgcggtt 120

agtaagggct atctatccgc cttaaggact ggctggtaca cttccgtcat tacgattgag 180

ctgagtaata tcaaggagaa caaatgtaac ggcacagatg ccaaggtgaa actgattaag 240

caggagcttg acaagtacaa gaatgccgtt accgagctgc agttgctgat gcagagcaca 300

ccagcgacca ataatcgggc aagacgcgaa cttcctcggt ttatgaacta taccttgaat 360

aatgctaaga agacaaacgt cacactgtca aagaaacgaa aacgtaggtt cctcggcttc 420

cttctcggag taggcagtgc catcgcaagt ggagtagcag tctccaaagt gttgcaccta 480

gagggagaag tgaacaagat caaatctgca ctcctgagca ccaacaaagc tgtcgtctct 540

ttatccaatg gcgtttcagt gctcacgtct aaggttctcg acctgaaaaa ctacatcgac 600

aaacagctcc ttcccatcgt caacaaacag agttgctcga tttctaacat cgagacagtt 660

atcgaattcc aacagaagaa caatagactt ctggagatca ctcgggagtt ttccgtaaat 720

gcaggtgtga ctactccggt ctcaacctac atgctgacta attccgagtt attgtcgcta 780

atcaacgata tgcctataac gaatgaccag aagaaactca tgagcaacaa cgtgcagatc 840

gtaaggcagc aatcctactc catcatgtcc ataatcaagg aagaggtgct ggcgtatgta 900

gtccagttgc cactgtatgg agtgatagac accccatgtt ggaagctcca tacgagcccc 960

ctgtgtacca ctaatacaaa ggaggggtct aacatatgcc ttacccggac tgatcgtggg 1020

tggtattgcg acaatgccgg ttcagtgtcg ttctttccac aagccgaaac atgtaaggtg 1080

cagtcaaacc gagtgttctg tgacacaatg aatagcttga cattgccctc tgaggtgaac 1140

ctgtgcaatg tggacatttt caaccccaag tacgactgca aaatcatgac aagcaaaact 1200

gacgtgtcta gctctgtcat tacctctcta ggagccattg tgagctgcta cggtaagaca 1260

aaatgcacag cttcaaacaa gaatagaggc atcatcaaga ccttctccaa tgggtgtgat 1320

tacgtgagca ataaaggtgt ggacaccgtt agcgtaggca ataccctgta ttacgttaat 1380

aagcaggaag gcaaaagtct gtacgtcaaa ggggagccca tcataaactt ctacgatcct 1440

ctcgtgtttc caagcgatga gtttgatgcc tccatttcac aggtgaacga aaagatcaac 1500

cagtctctgg cctttattcg caaaagtgat gaactgctgg gaagcgggta tattcccgaa 1560

gctcctaggg atggacaagc atatgtgcgc aaggatggtg aatgggtcct gctgtctacc 1620

tttctgttag ttccgagagg gagtcatcat caccaccatc actggtcaca ccctcagttt 1680

gagaaa 1686

<210> 6

<211> 488

<212>PRT

<213> Artificial sequence

<220>

<223> FH_50

<220>

<221> MISC_FEATURE

<222> (1)..(84)

<223> F2

<220>

<221> MISC_FEATURE

<222> (85)..(111)

<223> pep27

<220>

<221> MISC_FEATURE

<222> (112)..(488)

<223> extracellular F1

<400> 6

Gln Asn Ile Thr Glu Glu Phe Tyr Gln Ser Thr Cys Ser Ala Val Ser

1 5 10 15

Lys Gly Tyr Leu Ser Ala Leu Arg Thr Gly Trp Tyr Thr Ser Val Ile

20 25 30

Thr Ile Glu Leu Ser Asn Ile Lys Glu Asn Lys Cys Asn Gly Thr Asp

35 40 45

Ala Lys Val Lys Leu Ile Lys Gln Glu Leu Asp Lys Tyr Lys Asn Ala

50 55 60

Val Thr Glu Leu Gln Leu Leu Met Gln Ser Thr Pro Ala Thr Asn Asn

65 70 75 80

Arg Ala Arg Arg Glu Leu Pro Arg Phe Met Asn Tyr Thr Leu Asn Asn

85 90 95

Ala Lys Lys Thr Asn Val Thr Leu Ser Lys Lys Arg Lys Asn Asn Phe

100 105 110

Leu Gly Phe Cys Leu Gly Val Gly Ser Ala Ile Ala Ser Gly Val Ala

115 120 125

Val Ser Lys Val Leu His Leu Glu Gly Glu Val Asn Lys Ile Lys Ser

130 135 140

Ala Leu Leu Ser Thr Asn Lys Ala Val Val Ser Leu Ser Asn Gly Val

145 150 155 160

Ser Val Leu Val Val Lys Val Leu Asp Leu Lys Asn Tyr Ile Asp Lys

165 170 175

Gln Leu Leu Pro Ile Val Asn Lys Gln Ser Cys Ser Ile Ser Asn Ile

180 185 190

Glu Thr Val Ile Glu Phe Gln Gln Lys Asn Asn Arg Leu Leu Glu Ile

195 200 205

Thr Arg Glu Phe Ser Val Asn Ala Gly Val Thr Thr Pro Val Ser Thr

210 215 220

Tyr Met Leu Thr Asn Ser Glu Leu Leu Ser Leu Ile Asn Asp Met Pro

225 230 235 240

Ile Thr Asn Asp Gln Lys Lys Leu Met Ser Asn Asn Val Gln Ile Val

245 250 255

Arg Gln Gln Ser Tyr Ser Ile Met Ser Ile Ile Lys Glu Glu Val Leu

260 265 270

Ala Tyr Val Val Gln Leu Pro Leu Tyr Gly Val Ile Asp Thr Pro Cys

275 280 285

Trp Lys Leu His Thr Ser Pro Leu Cys Thr Thr Asn Thr Lys Glu Gly

290 295 300

Ser Asn Ile Cys Leu Thr Arg Thr Asp Arg Gly Trp Tyr Cys Asp Asn

305 310 315 320

Ala Gly Ser Val Ser Phe Phe Pro Gln Ala Glu Thr Cys Lys Val Gln

325 330 335

Ser Asn Arg Val Phe Cys Asp Thr Met Asn Ser Cys Thr Leu Pro Ser

340 345 350

Glu Val Asn Leu Cys Asn Val Asp Ile Phe Asn Pro Lys Tyr Asp Cys

355 360 365

Lys Ile Met Thr Ser Lys Thr Asp Val Ser Ser Ser Val Ile Thr Ser

370 375 380

Leu Gly Ala Ile Val Ser Cys Tyr Gly Lys Thr Lys Cys Thr Ala Ser

385 390 395 400

Asn Lys Asn Arg Gly Ile Ile Lys Thr Phe Ser Asn Gly Cys Asp Tyr

405 410 415

Val Ser Asn Lys Gly Val Asp Thr Val Ser Val Gly Asn Thr Leu Tyr

420 425 430

Tyr Val Asn Lys Gln Glu Gly Lys Ser Leu Tyr Val Lys Gly Glu Pro

435 440 445

Ile Ile Asn Phe Tyr Asp Pro Leu Val Phe Pro Ser Asp Glu Phe Asp

450 455 460

Ala Ser Ile Ser Gln Val Asn Glu Lys Ile Asn Gln Ser Leu Ala Phe

465 470 475 480

Ile Arg Lys Ser Asp Glu Leu Leu

485

<210> 7

<211> 488

<212>PRT

<213> Artificial sequence

<220>

<223> FH_81

<220>

<221> MISC_FEATURE

<222> (1)..(84)

<223> F2

<220>

<221> MISC_FEATURE

<222> (85)..(111)

<223> pep27

<220>

<221> MISC_FEATURE

<222> (112)..(488)

<223> extracellular F1

<400> 7

Gln Asn Ile Thr Glu Glu Phe Tyr Gln Ser Thr Cys Ser Ala Val Ser

1 5 10 15

Lys Gly Tyr Leu Ser Ala Leu Arg Thr Gly Trp Tyr Thr Ser Val Ile

20 25 30

Thr Ile Met Leu Ser Asn Ile Lys Glu Asn Lys Cys Asn Gly Thr Asp

35 40 45

Ala Lys Val Lys Leu Ile Lys Gln Glu Leu Asp Lys Tyr Lys Asn Ala

50 55 60

Val Thr Glu Leu Gln Leu Leu Met Gln Ser Thr Pro Ala Thr Asn Asn

65 70 75 80

Arg Ala Arg Arg Glu Leu Pro Arg Phe Met Asn Tyr Thr Leu Asn Asn

85 90 95

Ala Lys Lys Thr Asn Val Thr Leu Ser Lys Lys Arg Lys Asn Asn Phe

100 105 110

Leu Gly Phe Cys Leu Gly Val Gly Ser Ala Ile Ala Ser Gly Val Ala

115 120 125

Val Ser Lys Val Leu His Leu Glu Gly Glu Val Asn Lys Ile Lys Ser

130 135 140

Ala Leu Leu Ser Thr Asn Lys Ala Val Val Ser Leu Ser Asn Gly Val

145 150 155 160

Ser Val Leu Val Val Lys Val Leu Asp Leu Lys Asn Tyr Ile Asp Lys

165 170 175

Gln Leu Leu Pro Ile Val Asn Lys Gln Ser Cys Ser Ile Ser Asn Ile

180 185 190

Glu Thr Val Ile Glu Phe Gln Gln Lys Asn Asn Arg Leu Leu Glu Ile

195 200 205

Thr Arg Glu Phe Ser Val Asn Ala Gly Val Thr Thr Pro Val Ser Thr

210 215 220

Tyr Met Leu Thr Asn Ser Glu Leu Leu Ser Leu Ile Asn Asp Met Pro

225 230 235 240

Ile Thr Asn Asp Gln Lys Lys Leu Met Ser Asn Asn Val Gln Ile Val

245 250 255

Arg Gln Gln Ser Tyr Ser Ile Met Ser Ile Ile Lys Glu Glu Val Leu

260 265 270

Ala Tyr Val Val Gln Leu Pro Leu Tyr Gly Val Ile Asp Thr Pro Cys

275 280 285

Trp Lys Leu His Thr Ser Pro Leu Cys Thr Thr Asn Thr Lys Glu Gly

290 295 300

Ser Asn Ile Cys Leu Thr Arg Thr Asp Arg Gly Trp Tyr Cys Asp Asn

305 310 315 320

Ala Gly Ser Val Ser Phe Phe Pro Gln Ala Glu Thr Cys Lys Val Gln

325 330 335

Ser Asn Arg Val Phe Cys Asp Thr Met Asn Ser Cys Thr Leu Pro Ser

340 345 350

Glu Val Asn Leu Cys Asn Val Asp Ile Phe Asn Pro Lys Tyr Asp Cys

355 360 365

Lys Ile Met Thr Ser Lys Thr Asp Val Ser Ser Ser Val Ile Thr Ser

370 375 380

Leu Gly Ala Ile Val Ser Cys Tyr Gly Lys Thr Lys Cys Thr Ala Ser

385 390 395 400

Asn Lys Asn Arg Gly Ile Ile Lys Thr Phe Ser Asn Gly Cys Asp Tyr

405 410 415

Val Ser Asn Lys Gly Val Asp Thr Val Ser Val Gly Asn Thr Leu Tyr

420 425 430

Tyr Val Asn Lys Gln Glu Gly Lys Ser Leu Tyr Val Lys Gly Glu Pro

435 440 445

Ile Ile Asn Phe Tyr Asp Pro Leu Val Phe Pro Ser Asp Glu Phe Asp

450 455 460

Ala Ser Ile Ser Gln Val Asn Glu Lys Ile Asn Gln Ser Leu Ala Phe

465 470 475 480

Ile Arg Lys Ser Asp Glu Leu Leu

485

<210> 8

<211> 488

<212>PRT

<213> Artificial sequence

<220>

<223> FH_82

<220>

<221> MISC_FEATURE

<222> (1)..(84)

<223> F2

<220>

<221> MISC_FEATURE

<222> (85)..(111)

<223> pep27

<220>

<221> MISC_FEATURE

<222> (112)..(488)

<223> extracellular F1

<400> 8

Gln Asn Ile Thr Glu Glu Phe Tyr Gln Ser Thr Cys Ser Ala Val Ser

1 5 10 15

Arg Gly Tyr Leu Ser Ala Leu Arg Thr Gly Trp Tyr Thr Ser Val Ile

20 25 30

Thr Ile Glu Leu Ser Asn Ile Lys Glu Asn Lys Cys Asn Gly Thr Asp

35 40 45

Ala Lys Val Lys Leu Ile Lys Gln Glu Leu Asp Lys Tyr Lys Asn Ala

50 55 60

Val Thr Glu Leu Gln Leu Leu Met Gln Ser Thr Pro Ala Thr Asn Asn

65 70 75 80

Arg Ala Arg Arg Glu Leu Pro Arg Phe Met Asn Tyr Thr Leu Asn Asn

85 90 95

Ala Lys Lys Thr Asn Val Thr Leu Ser Lys Lys Arg Lys Asn Asn Phe

100 105 110

Leu Gly Phe Cys Leu Gly Val Gly Ser Ala Ile Ala Ser Gly Val Ala

115 120 125

Val Ser Lys Val Leu His Leu Glu Gly Glu Val Asn Lys Ile Lys Ser

130 135 140

Ala Leu Leu Ser Thr Asn Lys Ala Val Val Ser Leu Ser Asn Gly Val

145 150 155 160

Ser Val Leu Val Val Lys Val Leu Asp Leu Lys Asn Tyr Ile Asp Lys

165 170 175

Gln Leu Leu Pro Ile Val Asn Lys Gln Ser Cys Ser Ile Ser Asn Ile

180 185 190

Glu Thr Val Ile Glu Phe Gln Gln Lys Asn Asn Arg Leu Leu Glu Ile

195 200 205

Thr Arg Glu Phe Ser Val Asn Ala Gly Val Thr Thr Pro Val Ser Thr

210 215 220

Tyr Met Leu Thr Asn Ser Glu Leu Leu Ser Leu Ile Asn Asp Met Pro

225 230 235 240

Ile Thr Asn Asp Gln Lys Lys Leu Met Ser Asn Asn Val Gln Ile Val

245 250 255

Arg Gln Gln Ser Tyr Ser Ile Met Ser Ile Ile Lys Glu Glu Val Leu

260 265 270

Ala Tyr Val Val Gln Leu Pro Leu Tyr Gly Val Ile Asp Thr Pro Cys

275 280 285

Trp Lys Leu His Thr Ser Pro Leu Cys Thr Thr Asn Thr Lys Glu Gly

290 295 300

Ser Asn Ile Cys Leu Thr Arg Thr Asp Arg Gly Trp Tyr Cys Asp Asn

305 310 315 320

Ala Gly Ser Val Ser Phe Phe Pro Gln Ala Glu Thr Cys Lys Val Gln

325 330 335

Ser Asn Arg Val Phe Cys Asp Thr Met Asn Ser Cys Thr Leu Pro Ser

340 345 350

Glu Val Asn Leu Cys Asn Thr Asp Ile Phe Asn Pro Lys Tyr Asp Cys

355 360 365

Lys Ile Met Thr Ser Lys Thr Asp Val Ser Ser Ser Val Ile Thr Ser

370 375 380

Leu Gly Ala Ile Val Ser Cys Tyr Gly Lys Thr Lys Cys Thr Ala Ser

385 390 395 400

Asn Lys Asn Arg Gly Ile Ile Lys Thr Phe Ser Asn Gly Cys Asp Tyr

405 410 415

Val Ser Asn Lys Gly Val Asp Thr Val Ser Val Gly Asn Thr Leu Tyr

420 425 430

Tyr Val Asn Lys Gln Glu Gly Lys Ser Leu Tyr Val Lys Gly Glu Pro

435 440 445

Ile Ile Asn Phe Tyr Asp Pro Leu Val Phe Pro Ser Asp Glu Phe Asp

450 455 460

Ala Ser Ile Ser Gln Val Asn Glu Lys Ile Asn Gln Ser Leu Ala Phe

465 470 475 480

Ile Arg Lys Ser Asp Glu Leu Leu

485

<210> 9

<211> 488

<212>PRT

<213> Artificial sequence

<220>

<223> FH_85

<220>

<221> MISC_FEATURE

<222> (1)..(84)

<223> F2

<220>

<221> MISC_FEATURE

<222> (85)..(111)

<223> pep27

<220>

<221> MISC_FEATURE

<222> (112)..(488)

<223> extracellular F1 r

<400> 9

Gln Asn Ile Thr Glu Glu Phe Tyr Gln Ser Thr Cys Ser Ala Val Ser

1 5 10 15

Arg Gly Tyr Leu Ser Ala Leu Arg Thr Gly Trp Tyr Thr Ser Val Ile

20 25 30

Thr Ile Met Leu Ser Asn Ile Lys Glu Asn Lys Cys Asn Gly Thr Asp

35 40 45

Ala Lys Val Lys Leu Ile Lys Gln Glu Leu Asp Lys Tyr Lys Asn Ala

50 55 60

Val Thr Glu Leu Gln Leu Leu Met Gln Ser Thr Pro Ala Thr Asn Asn

65 70 75 80

Arg Ala Arg Arg Glu Leu Pro Arg Phe Met Asn Tyr Thr Leu Asn Asn

85 90 95

Ala Lys Lys Thr Asn Val Thr Leu Ser Lys Lys Arg Lys Asn Asn Phe

100 105 110

Leu Gly Phe Cys Leu Gly Val Gly Ser Ala Ile Ala Ser Gly Val Ala

115 120 125

Val Ser Lys Val Leu His Leu Glu Gly Glu Val Asn Lys Ile Lys Ser

130 135 140

Ala Leu Leu Ser Thr Asn Lys Ala Val Val Ser Leu Ser Asn Gly Val

145 150 155 160

Ser Val Leu Val Val Lys Val Leu Asp Leu Lys Asn Tyr Ile Asp Lys

165 170 175

Gln Leu Leu Pro Ile Val Asn Lys Gln Ser Cys Ser Ile Ser Asn Ile

180 185 190

Glu Thr Val Ile Glu Phe Gln Gln Lys Asn Asn Arg Leu Leu Glu Ile

195 200 205

Thr Arg Glu Phe Ser Val Asn Ala Gly Val Thr Thr Pro Val Ser Thr

210 215 220

Tyr Met Leu Thr Asn Ser Glu Leu Leu Ser Leu Ile Asn Asp Met Pro

225 230 235 240

Ile Thr Asn Asp Gln Lys Lys Leu Met Ser Asn Asn Val Gln Ile Val

245 250 255

Arg Gln Gln Ser Tyr Ser Ile Met Ser Ile Ile Lys Glu Glu Val Leu

260 265 270

Ala Tyr Val Val Gln Leu Pro Leu Tyr Gly Val Ile Asp Thr Pro Cys

275 280 285

Trp Lys Leu His Thr Ser Pro Leu Cys Thr Thr Asn Thr Lys Glu Gly

290 295 300

Ser Asn Ile Cys Leu Thr Arg Thr Asp Arg Gly Trp Tyr Cys Asp Asn

305 310 315 320

Ala Gly Ser Val Ser Phe Phe Pro Gln Ala Glu Thr Cys Lys Val Gln

325 330 335

Ser Asn Arg Val Phe Cys Asp Thr Met Asn Ser Cys Thr Leu Pro Ser

340 345 350

Glu Val Asn Leu Cys Asn Thr Asp Ile Phe Asn Pro Lys Tyr Asp Cys

355 360 365

Lys Ile Met Thr Ser Lys Thr Asp Val Ser Ser Ser Val Ile Thr Ser

370 375 380

Leu Gly Ala Ile Val Ser Cys Tyr Gly Lys Thr Lys Cys Thr Ala Ser

385 390 395 400

Asn Lys Asn Arg Gly Ile Ile Lys Thr Phe Ser Asn Gly Cys Asp Tyr

405 410 415

Val Ser Asn Lys Gly Val Asp Thr Val Ser Val Gly Asn Thr Leu Tyr

420 425 430

Tyr Val Asn Lys Gln Glu Gly Lys Ser Leu Tyr Val Lys Gly Glu Pro

435 440 445

Ile Ile Asn Phe Tyr Asp Pro Leu Val Phe Pro Ser Asp Glu Phe Asp

450 455 460

Ala Ser Ile Ser Gln Val Asn Glu Lys Ile Asn Gln Ser Leu Ala Phe

465 470 475 480

Ile Arg Lys Ser Asp Glu Leu Leu

485

<210> 10

<211> 517

<212>PRT

<213> Artificial sequence

<220>

<223> FH50-foldon-tag

<220>

<221> MISC_FEATURE

<222> (1)..(84)

<223> F2

<220>

<221> MISC_FEATURE

<222> (85)..(111)

<223> pep27

<220>

<221> MISC_FEATURE

<222> (112)..(488)

<223> extracellular F1 r

<220>

<221> MISC_FEATURE

<222> (489)..(517)

<223> foldon

<400> 10

Gln Asn Ile Thr Glu Glu Phe Tyr Gln Ser Thr Cys Ser Ala Val Ser

1 5 10 15

Lys Gly Tyr Leu Ser Ala Leu Arg Thr Gly Trp Tyr Thr Ser Val Ile

20 25 30

Thr Ile Glu Leu Ser Asn Ile Lys Glu Asn Lys Cys Asn Gly Thr Asp

35 40 45

Ala Lys Val Lys Leu Ile Lys Gln Glu Leu Asp Lys Tyr Lys Asn Ala

50 55 60

Val Thr Glu Leu Gln Leu Leu Met Gln Ser Thr Pro Ala Thr Asn Asn

65 70 75 80

Arg Ala Arg Arg Glu Leu Pro Arg Phe Met Asn Tyr Thr Leu Asn Asn

85 90 95

Ala Lys Lys Thr Asn Val Thr Leu Ser Lys Lys Arg Lys Asn Asn Phe

100 105 110

Leu Gly Phe Cys Leu Gly Val Gly Ser Ala Ile Ala Ser Gly Val Ala

115 120 125

Val Ser Lys Val Leu His Leu Glu Gly Glu Val Asn Lys Ile Lys Ser

130 135 140

Ala Leu Leu Ser Thr Asn Lys Ala Val Val Ser Leu Ser Asn Gly Val

145 150 155 160

Ser Val Leu Val Val Lys Val Leu Asp Leu Lys Asn Tyr Ile Asp Lys

165 170 175

Gln Leu Leu Pro Ile Val Asn Lys Gln Ser Cys Ser Ile Ser Asn Ile

180 185 190

Glu Thr Val Ile Glu Phe Gln Gln Lys Asn Asn Arg Leu Leu Glu Ile

195 200 205

Thr Arg Glu Phe Ser Val Asn Ala Gly Val Thr Thr Pro Val Ser Thr

210 215 220

Tyr Met Leu Thr Asn Ser Glu Leu Leu Ser Leu Ile Asn Asp Met Pro

225 230 235 240

Ile Thr Asn Asp Gln Lys Lys Leu Met Ser Asn Asn Val Gln Ile Val

245 250 255

Arg Gln Gln Ser Tyr Ser Ile Met Ser Ile Ile Lys Glu Glu Val Leu

260 265 270

Ala Tyr Val Val Gln Leu Pro Leu Tyr Gly Val Ile Asp Thr Pro Cys

275 280 285

Trp Lys Leu His Thr Ser Pro Leu Cys Thr Thr Asn Thr Lys Glu Gly

290 295 300

Ser Asn Ile Cys Leu Thr Arg Thr Asp Arg Gly Trp Tyr Cys Asp Asn

305 310 315 320

Ala Gly Ser Val Ser Phe Phe Pro Gln Ala Glu Thr Cys Lys Val Gln

325 330 335

Ser Asn Arg Val Phe Cys Asp Thr Met Asn Ser Cys Thr Leu Pro Ser

340 345 350

Glu Val Asn Leu Cys Asn Val Asp Ile Phe Asn Pro Lys Tyr Asp Cys

355 360 365

Lys Ile Met Thr Ser Lys Thr Asp Val Ser Ser Ser Val Ile Thr Ser

370 375 380

Leu Gly Ala Ile Val Ser Cys Tyr Gly Lys Thr Lys Cys Thr Ala Ser

385 390 395 400

Asn Lys Asn Arg Gly Ile Ile Lys Thr Phe Ser Asn Gly Cys Asp Tyr

405 410 415

Val Ser Asn Lys Gly Val Asp Thr Val Ser Val Gly Asn Thr Leu Tyr

420 425 430

Tyr Val Asn Lys Gln Glu Gly Lys Ser Leu Tyr Val Lys Gly Glu Pro

435 440 445

Ile Ile Asn Phe Tyr Asp Pro Leu Val Phe Pro Ser Asp Glu Phe Asp

450 455 460

Ala Ser Ile Ser Gln Val Asn Glu Lys Ile Asn Gln Ser Leu Ala Phe

465 470 475 480

Ile Arg Lys Ser Asp Glu Leu Leu Gly Ser Gly Tyr Ile Pro Glu Ala

485 490 495

Pro Arg Asp Gly Gln Ala Tyr Val Arg Lys Asp Gly Glu Trp Val Leu

500 505 510

Leu Ser Thr Phe Leu

515

<210> 11

<211> 517

<212>PRT

<213> Artificial sequence

<220>

<223> FH81-foldon-tag

<220>

<221> MISC_FEATURE

<222> (1)..(84)

<223> F2

<220>

<221> MISC_FEATURE

<222> (85)..(111)

<223> pep27

<220>

<221> MISC_FEATURE

<222> (112)..(488)

<223> extracellular F1

<220>

<221> MISC_FEATURE

<222> (489)..(517)

<223> foldon

<400> 11

Gln Asn Ile Thr Glu Glu Phe Tyr Gln Ser Thr Cys Ser Ala Val Ser

1 5 10 15

Lys Gly Tyr Leu Ser Ala Leu Arg Thr Gly Trp Tyr Thr Ser Val Ile

20 25 30

Thr Ile Met Leu Ser Asn Ile Lys Glu Asn Lys Cys Asn Gly Thr Asp

35 40 45

Ala Lys Val Lys Leu Ile Lys Gln Glu Leu Asp Lys Tyr Lys Asn Ala

50 55 60

Val Thr Glu Leu Gln Leu Leu Met Gln Ser Thr Pro Ala Thr Asn Asn

65 70 75 80

Arg Ala Arg Arg Glu Leu Pro Arg Phe Met Asn Tyr Thr Leu Asn Asn

85 90 95

Ala Lys Lys Thr Asn Val Thr Leu Ser Lys Lys Arg Lys Asn Asn Phe

100 105 110

Leu Gly Phe Cys Leu Gly Val Gly Ser Ala Ile Ala Ser Gly Val Ala

115 120 125

Val Ser Lys Val Leu His Leu Glu Gly Glu Val Asn Lys Ile Lys Ser

130 135 140

Ala Leu Leu Ser Thr Asn Lys Ala Val Val Ser Leu Ser Asn Gly Val

145 150 155 160

Ser Val Leu Val Val Lys Val Leu Asp Leu Lys Asn Tyr Ile Asp Lys

165 170 175

Gln Leu Leu Pro Ile Val Asn Lys Gln Ser Cys Ser Ile Ser Asn Ile

180 185 190

Glu Thr Val Ile Glu Phe Gln Gln Lys Asn Asn Arg Leu Leu Glu Ile

195 200 205

Thr Arg Glu Phe Ser Val Asn Ala Gly Val Thr Thr Pro Val Ser Thr

210 215 220

Tyr Met Leu Thr Asn Ser Glu Leu Leu Ser Leu Ile Asn Asp Met Pro

225 230 235 240

Ile Thr Asn Asp Gln Lys Lys Leu Met Ser Asn Asn Val Gln Ile Val

245 250 255

Arg Gln Gln Ser Tyr Ser Ile Met Ser Ile Ile Lys Glu Glu Val Leu

260 265 270

Ala Tyr Val Val Gln Leu Pro Leu Tyr Gly Val Ile Asp Thr Pro Cys

275 280 285

Trp Lys Leu His Thr Ser Pro Leu Cys Thr Thr Asn Thr Lys Glu Gly

290 295 300

Ser Asn Ile Cys Leu Thr Arg Thr Asp Arg Gly Trp Tyr Cys Asp Asn

305 310 315 320

Ala Gly Ser Val Ser Phe Phe Pro Gln Ala Glu Thr Cys Lys Val Gln

325 330 335

Ser Asn Arg Val Phe Cys Asp Thr Met Asn Ser Cys Thr Leu Pro Ser

340 345 350

Glu Val Asn Leu Cys Asn Val Asp Ile Phe Asn Pro Lys Tyr Asp Cys

355 360 365

Lys Ile Met Thr Ser Lys Thr Asp Val Ser Ser Ser Val Ile Thr Ser

370 375 380

Leu Gly Ala Ile Val Ser Cys Tyr Gly Lys Thr Lys Cys Thr Ala Ser

385 390 395 400

Asn Lys Asn Arg Gly Ile Ile Lys Thr Phe Ser Asn Gly Cys Asp Tyr

405 410 415

Val Ser Asn Lys Gly Val Asp Thr Val Ser Val Gly Asn Thr Leu Tyr

420 425 430

Tyr Val Asn Lys Gln Glu Gly Lys Ser Leu Tyr Val Lys Gly Glu Pro

435 440 445

Ile Ile Asn Phe Tyr Asp Pro Leu Val Phe Pro Ser Asp Glu Phe Asp

450 455 460

Ala Ser Ile Ser Gln Val Asn Glu Lys Ile Asn Gln Ser Leu Ala Phe

465 470 475 480

Ile Arg Lys Ser Asp Glu Leu Leu Gly Ser Gly Tyr Ile Pro Glu Ala

485 490 495

Pro Arg Asp Gly Gln Ala Tyr Val Arg Lys Asp Gly Glu Trp Val Leu

500 505 510

Leu Ser Thr Phe Leu

515

<210> 12

<211> 517

<212>PRT

<213> Artificial sequence

<220>

<223> FH82-foldon-tag

<220>

<221> MISC_FEATURE

<222> (1)..(84)

<223> F2

<220>

<221> MISC_FEATURE

<222> (85)..(111)

<223> pep27

<220>

<221> MISC_FEATURE

<222> (112)..(488)

<223> extracellular F1

<220>

<221> MISC_FEATURE

<222> (489)..(517)

<223> foldon

<400> 12

Gln Asn Ile Thr Glu Glu Phe Tyr Gln Ser Thr Cys Ser Ala Val Ser

1 5 10 15

Arg Gly Tyr Leu Ser Ala Leu Arg Thr Gly Trp Tyr Thr Ser Val Ile

20 25 30

Thr Ile Glu Leu Ser Asn Ile Lys Glu Asn Lys Cys Asn Gly Thr Asp

35 40 45

Ala Lys Val Lys Leu Ile Lys Gln Glu Leu Asp Lys Tyr Lys Asn Ala

50 55 60

Val Thr Glu Leu Gln Leu Leu Met Gln Ser Thr Pro Ala Thr Asn Asn

65 70 75 80

Arg Ala Arg Arg Glu Leu Pro Arg Phe Met Asn Tyr Thr Leu Asn Asn

85 90 95

Ala Lys Lys Thr Asn Val Thr Leu Ser Lys Lys Arg Lys Asn Asn Phe

100 105 110

Leu Gly Phe Cys Leu Gly Val Gly Ser Ala Ile Ala Ser Gly Val Ala

115 120 125

Val Ser Lys Val Leu His Leu Glu Gly Glu Val Asn Lys Ile Lys Ser

130 135 140

Ala Leu Leu Ser Thr Asn Lys Ala Val Val Ser Leu Ser Asn Gly Val

145 150 155 160

Ser Val Leu Val Val Lys Val Leu Asp Leu Lys Asn Tyr Ile Asp Lys

165 170 175

Gln Leu Leu Pro Ile Val Asn Lys Gln Ser Cys Ser Ile Ser Asn Ile

180 185 190

Glu Thr Val Ile Glu Phe Gln Gln Lys Asn Asn Arg Leu Leu Glu Ile

195 200 205

Thr Arg Glu Phe Ser Val Asn Ala Gly Val Thr Thr Pro Val Ser Thr

210 215 220

Tyr Met Leu Thr Asn Ser Glu Leu Leu Ser Leu Ile Asn Asp Met Pro

225 230 235 240

Ile Thr Asn Asp Gln Lys Lys Leu Met Ser Asn Asn Val Gln Ile Val

245 250 255

Arg Gln Gln Ser Tyr Ser Ile Met Ser Ile Ile Lys Glu Glu Val Leu

260 265 270

Ala Tyr Val Val Gln Leu Pro Leu Tyr Gly Val Ile Asp Thr Pro Cys

275 280 285

Trp Lys Leu His Thr Ser Pro Leu Cys Thr Thr Asn Thr Lys Glu Gly

290 295 300

Ser Asn Ile Cys Leu Thr Arg Thr Asp Arg Gly Trp Tyr Cys Asp Asn

305 310 315 320

Ala Gly Ser Val Ser Phe Phe Pro Gln Ala Glu Thr Cys Lys Val Gln

325 330 335

Ser Asn Arg Val Phe Cys Asp Thr Met Asn Ser Cys Thr Leu Pro Ser

340 345 350

Glu Val Asn Leu Cys Asn Thr Asp Ile Phe Asn Pro Lys Tyr Asp Cys

355 360 365

Lys Ile Met Thr Ser Lys Thr Asp Val Ser Ser Ser Val Ile Thr Ser

370 375 380

Leu Gly Ala Ile Val Ser Cys Tyr Gly Lys Thr Lys Cys Thr Ala Ser

385 390 395 400

Asn Lys Asn Arg Gly Ile Ile Lys Thr Phe Ser Asn Gly Cys Asp Tyr

405 410 415

Val Ser Asn Lys Gly Val Asp Thr Val Ser Val Gly Asn Thr Leu Tyr

420 425 430

Tyr Val Asn Lys Gln Glu Gly Lys Ser Leu Tyr Val Lys Gly Glu Pro

435 440 445

Ile Ile Asn Phe Tyr Asp Pro Leu Val Phe Pro Ser Asp Glu Phe Asp

450 455 460

Ala Ser Ile Ser Gln Val Asn Glu Lys Ile Asn Gln Ser Leu Ala Phe

465 470 475 480

Ile Arg Lys Ser Asp Glu Leu Leu Gly Ser Gly Tyr Ile Pro Glu Ala

485 490 495

Pro Arg Asp Gly Gln Ala Tyr Val Arg Lys Asp Gly Glu Trp Val Leu

500 505 510

Leu Ser Thr Phe Leu

515

<210> 13

<211> 517

<212>PRT

<213> Artificial sequence

<220>

<223> FH85-foldon-tag

<220>

<221> MISC_FEATURE

<222> (1)..(84)

<223> F2

<220>

<221> MISC_FEATURE

<222> (85)..(111)

<223> pep27

<220>

<221> MISC_FEATURE

<222> (112)..(488)

<223> extracellular F1

<220>

<221> MISC_FEATURE

<222> (489)..(517)

<223> foldon

<400> 13

Gln Asn Ile Thr Glu Glu Phe Tyr Gln Ser Thr Cys Ser Ala Val Ser

1 5 10 15

Arg Gly Tyr Leu Ser Ala Leu Arg Thr Gly Trp Tyr Thr Ser Val Ile

20 25 30

Thr Ile Met Leu Ser Asn Ile Lys Glu Asn Lys Cys Asn Gly Thr Asp

35 40 45

Ala Lys Val Lys Leu Ile Lys Gln Glu Leu Asp Lys Tyr Lys Asn Ala

50 55 60

Val Thr Glu Leu Gln Leu Leu Met Gln Ser Thr Pro Ala Thr Asn Asn

65 70 75 80

Arg Ala Arg Arg Glu Leu Pro Arg Phe Met Asn Tyr Thr Leu Asn Asn

85 90 95

Ala Lys Lys Thr Asn Val Thr Leu Ser Lys Lys Arg Lys Asn Asn Phe

100 105 110

Leu Gly Phe Cys Leu Gly Val Gly Ser Ala Ile Ala Ser Gly Val Ala

115 120 125

Val Ser Lys Val Leu His Leu Glu Gly Glu Val Asn Lys Ile Lys Ser

130 135 140

Ala Leu Leu Ser Thr Asn Lys Ala Val Val Ser Leu Ser Asn Gly Val

145 150 155 160

Ser Val Leu Val Val Lys Val Leu Asp Leu Lys Asn Tyr Ile Asp Lys

165 170 175

Gln Leu Leu Pro Ile Val Asn Lys Gln Ser Cys Ser Ile Ser Asn Ile

180 185 190

Glu Thr Val Ile Glu Phe Gln Gln Lys Asn Asn Arg Leu Leu Glu Ile

195 200 205

Thr Arg Glu Phe Ser Val Asn Ala Gly Val Thr Thr Pro Val Ser Thr

210 215 220

Tyr Met Leu Thr Asn Ser Glu Leu Leu Ser Leu Ile Asn Asp Met Pro

225 230 235 240

Ile Thr Asn Asp Gln Lys Lys Leu Met Ser Asn Asn Val Gln Ile Val

245 250 255

Arg Gln Gln Ser Tyr Ser Ile Met Ser Ile Ile Lys Glu Glu Val Leu

260 265 270

Ala Tyr Val Val Gln Leu Pro Leu Tyr Gly Val Ile Asp Thr Pro Cys

275 280 285

Trp Lys Leu His Thr Ser Pro Leu Cys Thr Thr Asn Thr Lys Glu Gly

290 295 300

Ser Asn Ile Cys Leu Thr Arg Thr Asp Arg Gly Trp Tyr Cys Asp Asn

305 310 315 320

Ala Gly Ser Val Ser Phe Phe Pro Gln Ala Glu Thr Cys Lys Val Gln

325 330 335

Ser Asn Arg Val Phe Cys Asp Thr Met Asn Ser Cys Thr Leu Pro Ser

340 345 350

Glu Val Asn Leu Cys Asn Thr Asp Ile Phe Asn Pro Lys Tyr Asp Cys

355 360 365

Lys Ile Met Thr Ser Lys Thr Asp Val Ser Ser Ser Val Ile Thr Ser

370 375 380

Leu Gly Ala Ile Val Ser Cys Tyr Gly Lys Thr Lys Cys Thr Ala Ser

385 390 395 400

Asn Lys Asn Arg Gly Ile Ile Lys Thr Phe Ser Asn Gly Cys Asp Tyr

405 410 415

Val Ser Asn Lys Gly Val Asp Thr Val Ser Val Gly Asn Thr Leu Tyr

420 425 430

Tyr Val Asn Lys Gln Glu Gly Lys Ser Leu Tyr Val Lys Gly Glu Pro

435 440 445

Ile Ile Asn Phe Tyr Asp Pro Leu Val Phe Pro Ser Asp Glu Phe Asp

450 455 460

Ala Ser Ile Ser Gln Val Asn Glu Lys Ile Asn Gln Ser Leu Ala Phe

465 470 475 480

Ile Arg Lys Ser Asp Glu Leu Leu Gly Ser Gly Tyr Ile Pro Glu Ala

485 490 495

Pro Arg Asp Gly Gln Ala Tyr Val Arg Lys Asp Gly Glu Trp Val Leu

500 505 510

Leu Ser Thr Phe Leu

515

<210> 14

<211> 1626

<212> DNA

<213> Artificial sequence

<220>

<223> Sequence encoding the FH50 foldon tag

<400> 14

atggaactgc tgatacttaa ggctaacgcg ataactacga tcctcaccgc cgtgaccttt 60

tgctttgcct ctggccaaaa cattactgag gaattctacc agtcaacgtg cagtgcagtg 120

agcaaagggt atctgtccgc cctgagaacc gggtggtata cttccgtcat taccatcgag 180

ctgtccaaca taaaagagaa taagtgcaac ggcaccgatg ctaaagtgaa actgatcaaa 240

caggaactcg ataaatacaa gaatgcagtt acagagcttc agctcctgat gcagagcact 300

ccggccacca ataatagggc aaggagagaa ttgccacgat ttatgaatta cacactcaac 360

aacgcgaaga aaactaacgt gactctgtcc aagaaacgta agaataactt cttggggttc 420

tgtctgggtg taggtagcgc cattgcttct ggggtggccg tcagcaaagt gcttcacctg 480

gaaggagagg tgaacaagat caagtctgca ctgctgtcta caaacaaagc agtggtgagc 540

ctgtccaacg gagtatccgt tctggtggtc aaagtcctgg atctgaagaa ttatatcgac 600

aaacaactgc tccccattgt gaacaagcag agttgttcaa tcagcaacat agaaactgtg 660

attgagttcc aacagaagaa caataggctg ctcgaaatta ccagagagtt tagcgtcaat 720

gctggtgtca caaccccagt cagcacttac atgctgacta attccgagtt gcttagcctt 780

attaacgaca tgcctatcac caatgaccag aagaagctga tgagtaataa tgtgcagatt 840

gtgcgccagc agagttacag cattatgagt attatcaaag aggaggtatt ggcttatgtg 900

gttcagcttc cgctgtatgg ggtcatcgac acaccttgtt ggaagttgca taccagtccc 960

ctgtgtacga caaacaccaa ggaaggtagt aacatctgct tgacacgtac cgatcggggt 1020

tggtattgcg ataacgccgg gtctgttagt ttctttcctc aagccgagac atgcaaagtc 1080

cagagcaatc gcgtgttctg tgacacgatg aacagctgta ctttgccatc agaggttaat 1140

ctgtgcaatg tagacatctt caaccccaaa tacgactgta agatcatgac cagcaagact 1200

gatgtcagct cctccgttat aacatcactc ggcgctatcg tgtcttgcta tggcaagacc 1260

aagtgtacag cgtccaataa gaatcggggc attatcaaga cattctccaa cggatgtgac 1320

tacgtgagca acaaaggagt ggacaccgtg tcagtcggaa atacactgta ttacgtgaat 1380

aagcaggagg gcaaatctct ttacgtgaag ggcgaaccaa tcatcaactt ctatgatccc 1440

ctcgtctttc cttctgatga gtttgacgcc tctatttctc aggttaacga gaagatcaat 1500

cagtctctgg cctttatacg caaaagcgat gaactcctgg gatcaggcta cattcccgaa 1560

gctccaaggg acggacaagc atacgtacga aaagatggcg aatgggtact cctctcaacg 1620

tttctg 1626

<210> 15

<211> 1626

<212> DNA

<213> Artificial sequence

<220>

<223> Sequence encoding FH81 foldon tag

<400> 15

atggaactgc tgatacttaa ggctaacgcg ataactacga tcctcaccgc cgtgaccttt 60

tgctttgcct ctggccaaaa cattactgag gaattctacc agtcaacgtg cagtgcagtg 120

agcaaagggt atctgtccgc cctgagaacc gggtggtata cttccgtcat taccatcatg 180

ctgtccaaca taaaagagaa taagtgcaac ggcaccgatg ctaaagtgaa actgatcaaa 240

caggaactcg ataaatacaa gaatgcagtt acagagcttc agctcctgat gcagagcact 300

ccggccacca ataatagggc aaggagagaa ttgccacgat ttatgaatta cacactcaac 360

aacgcgaaga aaactaacgt gactctgtcc aagaaacgta agaataactt cttggggttc 420

tgtctgggtg taggtagcgc cattgcttct ggggtggccg tcagcaaagt gcttcacctg 480

gaaggagagg tgaacaagat caagtctgca ctgctgtcta caaacaaagc agtggtgagc 540

ctgtccaacg gagtatccgt tctggtggtc aaagtcctgg atctgaagaa ttatatcgac 600

aaacaactgc tccccattgt gaacaagcag agttgttcaa tcagcaacat agaaactgtg 660

attgagttcc aacagaagaa caataggctg ctcgaaatta ccagagagtt tagcgtcaat 720

gctggtgtca caaccccagt cagcacttac atgctgacta attccgagtt gcttagcctt 780

attaacgaca tgcctatcac caatgaccag aagaagctga tgagtaataa tgtgcagatt 840

gtgcgccagc agagttacag cattatgagt attatcaaag aggaggtatt ggcttatgtg 900

gttcagcttc cgctgtatgg ggtcatcgac acaccttgtt ggaagttgca taccagtccc 960

ctgtgtacga caaacaccaa ggaaggtagt aacatctgct tgacacgtac cgatcggggt 1020

tggtattgcg ataacgccgg gtctgttagt ttctttcctc aagccgagac atgcaaagtc 1080

cagagcaatc gcgtgttctg tgacacgatg aacagctgta ctttgccatc agaggttaat 1140

ctgtgcaatg tagacatctt caaccccaaa tacgactgta agatcatgac cagcaagact 1200

gatgtcagct cctccgttat aacatcactc ggcgctatcg tgtcttgcta tggcaagacc 1260

aagtgtacag cgtccaataa gaatcggggc attatcaaga cattctccaa cggatgtgac 1320

tacgtgagca acaaaggagt ggacaccgtg tcagtcggaa atacactgta ttacgtgaat 1380

aagcaggagg gcaaatctct ttacgtgaag ggcgaaccaa tcatcaactt ctatgatccc 1440

ctcgtctttc cttctgatga gtttgacgcc tctatttctc aggttaacga gaagatcaat 1500

cagtctctgg cctttatacg caaaagcgat gaactcctgg gatcaggcta cattcccgaa 1560

gctccaaggg acggacaagc atacgtacga aaagatggcg aatgggtact cctctcaacg 1620

tttctg 1626

<210> 16

<211> 1626

<212> DNA

<213> Artificial sequence

<220>

<223> Sequence encoding FH82 foldon tag

<400> 16

atggaactgc tgatacttaa ggctaacgcg ataactacga tcctcaccgc cgtgaccttt 60

tgctttgcct ctggccaaaa cattactgag gaattctacc agtcaacgtg cagtgcagtg 120

agccgaggt atctgtccgc cctgagaacc gggtggtata cttccgtcat taccatcgag 180

ctgtccaaca taaaagagaa taagtgcaac ggcaccgatg ctaaagtgaa actgatcaaa 240

caggaactcg ataaatacaa gaatgcagtt acagagcttc agctcctgat gcagagcact 300

ccggccacca ataatagggc aaggagagaa ttgccacgat ttatgaatta cacactcaac 360

aacgcgaaga aaactaacgt gactctgtcc aagaaacgta agaataactt cttggggttc 420

tgtctgggtg taggtagcgc cattgcttct ggggtggccg tcagcaaagt gcttcacctg 480

gaaggagagg tgaacaagat caagtctgca ctgctgtcta caaacaaagc agtggtgagc 540

ctgtccaacg gagtatccgt tctggtggtc aaagtcctgg atctgaagaa ttatatcgac 600

aaacaactgc tccccattgt gaacaagcag agttgttcaa tcagcaacat agaaactgtg 660

attgagttcc aacagaagaa caataggctg ctcgaaatta ccagagagtt tagcgtcaat 720

gctggtgtca caaccccagt cagcacttac atgctgacta attccgagtt gcttagcctt 780

attaacgaca tgcctatcac caatgaccag aagaagctga tgagtaataa tgtgcagatt 840

gtgcgccagc agagttacag cattatgagt attatcaaag aggaggtatt ggcttatgtg 900

gttcagcttc cgctgtatgg ggtcatcgac acaccttgtt ggaagttgca taccagtccc 960

ctgtgtacga caaacaccaa ggaaggtagt aacatctgct tgacacgtac cgatcggggt 1020

tggtattgcg ataacgccgg gtctgttagt ttctttcctc aagccgagac atgcaaagtc 1080

cagagcaatc gcgtgttctg tgacacgatg aacagctgta ctttgccatc agaggttaat 1140

ctgtgcaata ccgacatctt caaccccaaa tacgactgta agatcatgac cagcaagact 1200

gatgtcagct cctccgttat aacatcactc ggcgctatcg tgtcttgcta tggcaagacc 1260

aagtgtacag cgtccaataa gaatcggggc attatcaaga cattctccaa cggatgtgac 1320

tacgtgagca acaaaggagt ggacaccgtg tcagtcggaa atacactgta ttacgtgaat 1380

aagcaggagg gcaaatctct ttacgtgaag ggcgaaccaa tcatcaactt ctatgatccc 1440

ctcgtctttc cttctgatga gtttgacgcc tctatttctc aggttaacga gaagatcaat 1500

cagtctctgg cctttatacg caaaagcgat gaactcctgg gatcaggcta cattcccgaa 1560

gctccaaggg acggacaagc atacgtacga aaagatggcg aatgggtact cctctcaacg 1620

tttctg 1626

<210> 17

<211> 1626

<212> DNA

<213> Artificial sequence

<220>

<223> Sequence encoding FH85 foldon tag

<400> 17

atggaactgc tgatacttaa ggctaacgcg ataactacga tcctcaccgc cgtgaccttt 60

tgctttgcct ctggccaaaa cattactgag gaattctacc agtcaacgtg cagtgcagtg 120

agccgaggt atctgtccgc cctgagaacc gggtggtata cttccgtcat taccatcatg 180

ctgtccaaca taaaagagaa taagtgcaac ggcaccgatg ctaaagtgaa actgatcaaa 240

caggaactcg ataaatacaa gaatgcagtt acagagcttc agctcctgat gcagagcact 300

ccggccacca ataatagggc aaggagagaa ttgccacgat ttatgaatta cacactcaac 360

aacgcgaaga aaactaacgt gactctgtcc aagaaacgta agaataactt cttggggttc 420

tgtctgggtg taggtagcgc cattgcttct ggggtggccg tcagcaaagt gcttcacctg 480

gaaggagagg tgaacaagat caagtctgca ctgctgtcta caaacaaagc agtggtgagc 540

ctgtccaacg gagtatccgt tctggtggtc aaagtcctgg atctgaagaa ttatatcgac 600

aaacaactgc tccccattgt gaacaagcag agttgttcaa tcagcaacat agaaactgtg 660

attgagttcc aacagaagaa caataggctg ctcgaaatta ccagagagtt tagcgtcaat 720

gctggtgtca caaccccagt cagcacttac atgctgacta attccgagtt gcttagcctt 780

attaacgaca tgcctatcac caatgaccag aagaagctga tgagtaataa tgtgcagatt 840

gtgcgccagc agagttacag cattatgagt attatcaaag aggaggtatt ggcttatgtg 900

gttcagcttc cgctgtatgg ggtcatcgac acaccttgtt ggaagttgca taccagtccc 960

ctgtgtacga caaacaccaa ggaaggtagt aacatctgct tgacacgtac cgatcggggt 1020

tggtattgcg ataacgccgg gtctgttagt ttctttcctc aagccgagac atgcaaagtc 1080

cagagcaatc gcgtgttctg tgacacgatg aacagctgta ctttgccatc agaggttaat 1140

ctgtgcaata ccgacatctt caaccccaaa tacgactgta agatcatgac cagcaagact 1200

gatgtcagct cctccgttat aacatcactc ggcgctatcg tgtcttgcta tggcaagacc 1260

aagtgtacag cgtccaataa gaatcggggc attatcaaga cattctccaa cggatgtgac 1320

tacgtgagca acaaaggagt ggacaccgtg tcagtcggaa atacactgta ttacgtgaat 1380

aagcaggagg gcaaatctct ttacgtgaag ggcgaaccaa tcatcaactt ctatgatccc 1440

ctcgtctttc cttctgatga gtttgacgcc tctatttctc aggttaacga gaagatcaat 1500

cagtctctgg cctttatacg caaaagcgat gaactcctgg gatcaggcta cattcccgaa 1560

gctccaaggg acggacaagc atacgtacga aaagatggcg aatgggtact cctctcaacg 1620

tttctg 1626

<210> 18

<211> 6

<212>PRT

<213> Artificial sequence

<220>

<223> GS linker

<400> 18

Gly Ser Gly Ser Gly Ser

15

<210> 19

<211> 10

<212>PRT

<213> Artificial sequence

<220>

<223> GS linker

<400> 19

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

1 5 10

<210> 20

<211> 15

<212>PRT

<213> Artificial sequence

<220>

<223> GS linker

<400> 20

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

1 5 10 15

<210> 21

<211> 29

<212>PRT

<213> Artificial sequence

<220>

<223> foldon

<400> 21

Gly Ser Gly Tyr Ile Pro Glu Ala Pro Arg Asp Gly Gln Ala Tyr Val

1 5 10 15

Arg Lys Asp Gly Glu Trp Val Leu Leu Ser Thr Phe Leu

20 25

<210> 22

<211> 20

<212>PRT

<213> Artificial sequence

<220>

<223> label

<400> 22

Leu Val Pro Arg Gly Ser His His His His His Trp Ser His Pro

1 5 10 15

Gln Phe Glu Lys

20

<210> 23

<211> 562

<212>PRT

<213> Artificial sequence

<220>

<223>DS-Cav1

<400> 23

Met Glu Leu Leu Ile Leu Lys Ala Asn Ala Ile Thr Thr Ile Leu Thr

1 5 10 15

Ala Val Thr Phe Cys Phe Ala Ser Gly Gln Asn Ile Thr Glu Glu Phe

20 25 30

Tyr Gln Ser Thr Cys Ser Ala Val Ser Lys Gly Tyr Leu Ser Ala Leu

35 40 45

Arg Thr Gly Trp Tyr Thr Ser Val Ile Thr Ile Glu Leu Ser Asn Ile

50 55 60

Lys Glu Asn Lys Cys Asn Gly Thr Asp Ala Lys Val Lys Leu Ile Lys

65 70 75 80

Gln Glu Leu Asp Lys Tyr Lys Asn Ala Val Thr Glu Leu Gln Leu Leu

85 90 95

Met Gln Ser Thr Pro Ala Thr Asn Asn Arg Ala Arg Arg Glu Leu Pro

100 105 110

Arg Phe Met Asn Tyr Thr Leu Asn Asn Ala Lys Lys Thr Asn Val Thr

115 120 125

Leu Ser Lys Lys Arg Lys Arg Arg Phe Leu Gly Phe Leu Leu Gly Val

130 135 140

Gly Ser Ala Ile Ala Ser Gly Val Ala Val Cys Lys Val Leu His Leu

145 150 155 160

Glu Gly Glu Val Asn Lys Ile Lys Ser Ala Leu Leu Ser Thr Asn Lys

165 170 175

Ala Val Val Ser Leu Ser Asn Gly Val Ser Val Leu Thr Phe Lys Val

180 185 190

Leu Asp Leu Lys Asn Tyr Ile Asp Lys Gln Leu Leu Pro Ile Leu Asn

195 200 205

Lys Gln Ser Cys Ser Ile Ser Asn Ile Glu Thr Val Ile Glu Phe Gln

210 215 220

Gln Lys Asn Asn Arg Leu Leu Glu Ile Thr Arg Glu Phe Ser Val Asn

225 230 235 240

Ala Gly Val Thr Thr Pro Val Ser Thr Tyr Met Leu Thr Asn Ser Glu

245 250 255

Leu Leu Ser Leu Ile Asn Asp Met Pro Ile Thr Asn Asp Gln Lys Lys

260 265 270

Leu Met Ser Asn Asn Val Gln Ile Val Arg Gln Gln Ser Tyr Ser Ile

275 280 285

Met Cys Ile Ile Lys Glu Glu Val Leu Ala Tyr Val Val Gln Leu Pro

290 295 300

Leu Tyr Gly Val Ile Asp Thr Pro Cys Trp Lys Leu His Thr Ser Pro

305 310 315 320

Leu Cys Thr Thr Asn Thr Lys Glu Gly Ser Asn Ile Cys Leu Thr Arg

325 330 335

Thr Asp Arg Gly Trp Tyr Cys Asp Asn Ala Gly Ser Val Ser Phe Phe

340 345 350

Pro Gln Ala Glu Thr Cys Lys Val Gln Ser Asn Arg Val Phe Cys Asp

355 360 365

Thr Met Asn Ser Leu Thr Leu Pro Ser Glu Val Asn Leu Cys Asn Val

370 375 380

Asp Ile Phe Asn Pro Lys Tyr Asp Cys Lys Ile Met Thr Ser Lys Thr

385 390 395 400

Asp Val Ser Ser Ser Val Ile Thr Ser Leu Gly Ala Ile Val Ser Cys

405 410 415

Tyr Gly Lys Thr Lys Cys Thr Ala Ser Asn Lys Asn Arg Gly Ile Ile

420 425 430

Lys Thr Phe Ser Asn Gly Cys Asp Tyr Val Ser Asn Lys Gly Val Asp

435 440 445

Thr Val Ser Val Gly Asn Thr Leu Tyr Tyr Val Asn Lys Gln Glu Gly

450 455 460

Lys Ser Leu Tyr Val Lys Gly Glu Pro Ile Ile Asn Phe Tyr Asp Pro

465 470 475 480

Leu Val Phe Pro Ser Asp Glu Phe Asp Ala Ser Ile Ser Gln Val Asn

485 490 495

Glu Lys Ile Asn Gln Ser Leu Ala Phe Ile Arg Lys Ser Asp Glu Leu

500 505 510

Leu Gly Ser Gly Tyr Ile Pro Glu Ala Pro Arg Asp Gly Gln Ala Tyr

515 520 525

Val Arg Lys Asp Gly Glu Trp Val Leu Leu Ser Thr Phe Leu Leu Val

530 535 540

Pro Arg Gly Ser His His His His His Trp Ser His Pro Gln Phe

545 550 555 560

Glu Lys

<210> 24

<211> 521

<212>PRT

<213> Artificial sequence

<220>

<223> delta FP furinwt Fecto

<400> 24

Met Glu Leu Leu Ile Leu Lys Ala Asn Ala Ile Thr Thr Ile Leu Thr

1 5 10 15

Ala Val Thr Phe Cys Phe Ala Ser Gly Gln Asn Ile Thr Glu Glu Phe

20 25 30

Tyr Gln Ser Thr Cys Ser Ala Val Ser Lys Gly Tyr Leu Ser Ala Leu

35 40 45

Arg Thr Gly Trp Tyr Thr Ser Val Ile Thr Ile Glu Leu Ser Asn Ile

50 55 60

Lys Glu Asn Lys Cys Asn Gly Thr Asp Ala Lys Val Lys Leu Ile Lys

65 70 75 80

Gln Glu Leu Asp Lys Tyr Lys Asn Ala Val Thr Glu Leu Gln Leu Leu

85 90 95

Met Gln Ser Thr Pro Ala Thr Asn Asn Arg Ala Arg Arg Glu Leu Pro

100 105 110

Arg Phe Met Asn Tyr Thr Leu Asn Asn Ala Lys Lys Thr Asn Val Thr

115 120 125

Leu Ser Lys Lys Arg Lys Arg Arg Ser Ala Ile Ala Ser Gly Val Ala

130 135 140

Val Ser Lys Val Leu His Leu Glu Gly Glu Val Asn Lys Ile Lys Ser

145 150 155 160

Ala Leu Leu Ser Thr Asn Lys Ala Val Val Ser Leu Ser Asn Gly Val

165 170 175

Ser Val Leu Thr Ser Lys Val Leu Asp Leu Lys Asn Tyr Ile Asp Lys

180 185 190

Gln Leu Leu Pro Ile Val Asn Lys Gln Ser Cys Ser Ile Ser Asn Ile

195 200 205

Glu Thr Val Ile Glu Phe Gln Gln Lys Asn Asn Arg Leu Leu Glu Ile

210 215 220

Thr Arg Glu Phe Ser Val Asn Ala Gly Val Thr Thr Pro Val Ser Thr

225 230 235 240

Tyr Met Leu Thr Asn Ser Glu Leu Leu Ser Leu Ile Asn Asp Met Pro

245 250 255

Ile Thr Asn Asp Gln Lys Lys Leu Met Ser Asn Asn Val Gln Ile Val

260 265 270

Arg Gln Gln Ser Tyr Ser Ile Met Ser Ile Ile Lys Glu Glu Val Leu

275 280 285

Ala Tyr Val Val Gln Leu Pro Leu Tyr Gly Val Ile Asp Thr Pro Cys

290 295 300

Trp Lys Leu His Thr Ser Pro Leu Cys Thr Thr Asn Thr Lys Glu Gly

305 310 315 320

Ser Asn Ile Cys Leu Thr Arg Thr Asp Arg Gly Trp Tyr Cys Asp Asn

325 330 335

Ala Gly Ser Val Ser Phe Phe Pro Gln Ala Glu Thr Cys Lys Val Gln

340 345 350

Ser Asn Arg Val Phe Cys Asp Thr Met Asn Ser Leu Thr Leu Pro Ser

355 360 365

Glu Val Asn Leu Cys Asn Val Asp Ile Phe Asn Pro Lys Tyr Asp Cys

370 375 380

Lys Ile Met Thr Ser Lys Thr Asp Val Ser Ser Ser Val Ile Thr Ser

385 390 395 400

Leu Gly Ala Ile Val Ser Cys Tyr Gly Lys Thr Lys Cys Thr Ala Ser

405 410 415

Asn Lys Asn Arg Gly Ile Ile Lys Thr Phe Ser Asn Gly Cys Asp Tyr

420 425 430

Val Ser Asn Lys Gly Val Asp Thr Val Ser Val Gly Asn Thr Leu Tyr

435 440 445

Tyr Val Asn Lys Gln Glu Gly Lys Ser Leu Tyr Val Lys Gly Glu Pro

450 455 460

Ile Ile Asn Phe Tyr Asp Pro Leu Val Phe Pro Ser Asp Glu Phe Asp

465 470 475 480

Ala Ser Ile Ser Gln Val Asn Glu Lys Ile Asn Gln Ser Leu Ala Phe

485 490 495

Ile Arg Lys Ser Asp Glu Leu Leu His Asn Val Asn Ala Gly Lys Ser

500 505 510

Thr Thr Asn His His His His His

515 520

<210> 25

<211> 6

<212>PRT

<213> Artificial sequence

<220>

<223> His tag

<400> 25

His His His His His

15

<210> 26

<211> 8

<212>PRT

<213> Artificial sequence

<220>

<223> Strep mark II

<400> 26

Trp Ser His Pro Gln Phe Glu Lys

15

<210> 27

<211> 5

<212>PRT

<213> Artificial sequence

<220>

<223> GS linker

<400> 27

Gly Gly Gly Gly Ser

15

<210> 28

<211> 8

<212>PRT

<213> Artificial sequence

<220>

<223> FLAG tag

<400> 28

Asp Tyr Lys Asp Asp Asp Asp Lys

15

<210> 29

<211> 11

<212>PRT

<213> Artificial sequence

<220>

<223> GGS+Flag

<400> 29

Gly Gly Ser Asp Tyr Lys Asp Asp Asp Asp Lys

1 5 10

<210> 30

<211> 227

<212>PRT

<213> Artificial sequence

<220>

<223>Fc

<400> 30

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly

1 5 10 15

Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

20 25 30

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

35 40 45

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

50 55 60

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

65 70 75 80

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

85 90 95

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

100 105 110

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

115 120 125

Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser

130 135 140

Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

145 150 155 160

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

165 170 175

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

180 185 190

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

195 200 205

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

210 215 220

Pro Gly Lys

225

<210> 31

<211> 769

<212>PRT

<213> Artificial sequence

<220>

<223> F_DS-Cav1-Fc

<400> 31

Met Glu Leu Leu Ile Leu Lys Ala Asn Ala Ile Thr Thr Ile Leu Thr

1 5 10 15

Ala Val Thr Phe Cys Phe Ala Ser Gly Gln Asn Ile Thr Glu Glu Phe

20 25 30

Tyr Gln Ser Thr Cys Ser Ala Val Ser Lys Gly Tyr Leu Ser Ala Leu

35 40 45

Arg Thr Gly Trp Tyr Thr Ser Val Ile Thr Ile Glu Leu Ser Asn Ile

50 55 60

Lys Glu Asn Lys Cys Asn Gly Thr Asp Ala Lys Val Lys Leu Ile Lys

65 70 75 80

Gln Glu Leu Asp Lys Tyr Lys Asn Ala Val Thr Glu Leu Gln Leu Leu

85 90 95

Met Gln Ser Thr Pro Ala Thr Asn Asn Arg Ala Arg Arg Glu Leu Pro

100 105 110

Arg Phe Met Asn Tyr Thr Leu Asn Asn Ala Lys Lys Thr Asn Val Thr

115 120 125

Leu Ser Lys Lys Arg Lys Arg Arg Phe Leu Gly Phe Leu Leu Gly Val

130 135 140

Gly Ser Ala Ile Ala Ser Gly Val Ala Val Cys Lys Val Leu His Leu

145 150 155 160

Glu Gly Glu Val Asn Lys Ile Lys Ser Ala Leu Leu Ser Thr Asn Lys

165 170 175

Ala Val Val Ser Leu Ser Asn Gly Val Ser Val Leu Thr Phe Lys Val

180 185 190

Leu Asp Leu Lys Asn Tyr Ile Asp Lys Gln Leu Leu Pro Ile Leu Asn

195 200 205

Lys Gln Ser Cys Ser Ile Ser Asn Ile Glu Thr Val Ile Glu Phe Gln

210 215 220

Gln Lys Asn Asn Arg Leu Leu Glu Ile Thr Arg Glu Phe Ser Val Asn

225 230 235 240

Ala Gly Val Thr Thr Pro Val Ser Thr Tyr Met Leu Thr Asn Ser Glu

245 250 255

Leu Leu Ser Leu Ile Asn Asp Met Pro Ile Thr Asn Asp Gln Lys Lys

260 265 270

Leu Met Ser Asn Asn Val Gln Ile Val Arg Gln Gln Ser Tyr Ser Ile

275 280 285

Met Cys Ile Ile Lys Glu Glu Val Leu Ala Tyr Val Val Gln Leu Pro

290 295 300

Leu Tyr Gly Val Ile Asp Thr Pro Cys Trp Lys Leu His Thr Ser Pro

305 310 315 320

Leu Cys Thr Thr Asn Thr Lys Glu Gly Ser Asn Ile Cys Leu Thr Arg

325 330 335

Thr Asp Arg Gly Trp Tyr Cys Asp Asn Ala Gly Ser Val Ser Phe Phe

340 345 350

Pro Gln Ala Glu Thr Cys Lys Val Gln Ser Asn Arg Val Phe Cys Asp

355 360 365

Thr Met Asn Ser Leu Thr Leu Pro Ser Glu Val Asn Leu Cys Asn Val

370 375 380

Asp Ile Phe Asn Pro Lys Tyr Asp Cys Lys Ile Met Thr Ser Lys Thr

385 390 395 400

Asp Val Ser Ser Ser Val Ile Thr Ser Leu Gly Ala Ile Val Ser Cys

405 410 415

Tyr Gly Lys Thr Lys Cys Thr Ala Ser Asn Lys Asn Arg Gly Ile Ile

420 425 430

Lys Thr Phe Ser Asn Gly Cys Asp Tyr Val Ser Asn Lys Gly Val Asp

435 440 445

Thr Val Ser Val Gly Asn Thr Leu Tyr Tyr Val Asn Lys Gln Glu Gly

450 455 460

Lys Ser Leu Tyr Val Lys Gly Glu Pro Ile Ile Asn Phe Tyr Asp Pro

465 470 475 480

Leu Val Phe Pro Ser Asp Glu Phe Asp Ala Ser Ile Ser Gln Val Asn

485 490 495

Glu Lys Ile Asn Gln Ser Leu Ala Phe Ile Arg Lys Ser Asp Glu Leu

500 505 510

Leu Gly Ser Gly Tyr Ile Pro Glu Ala Pro Arg Asp Gly Gln Ala Tyr

515 520 525

Val Arg Lys Asp Gly Glu Trp Val Leu Leu Ser Thr Phe Leu Asp Lys

530 535 540

Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro

545 550 555 560

Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser

565 570 575

Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp

580 585 590

Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn

595 600 605

Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val

610 615 620

Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu

625 630 635 640

Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys

645 650 655

Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr

660 665 670

Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr

675 680 685

Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu

690 695 700

Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu

705 710 715 720

Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys

725 730 735

Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu

740 745 750

Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

755 760 765

Lys

<210> 32

<211> 769

<212>PRT

<213> Artificial sequence

<220>

<223> FH_82-Fc

<400> 32

Met Glu Leu Leu Ile Leu Lys Ala Asn Ala Ile Thr Thr Ile Leu Thr

1 5 10 15

Ala Val Thr Phe Cys Phe Ala Ser Gly Gln Asn Ile Thr Glu Glu Phe

20 25 30

Tyr Gln Ser Thr Cys Ser Ala Val Ser Arg Gly Tyr Leu Ser Ala Leu

35 40 45

Arg Thr Gly Trp Tyr Thr Ser Val Ile Thr Ile Glu Leu Ser Asn Ile

50 55 60

Lys Glu Asn Lys Cys Asn Gly Thr Asp Ala Lys Val Lys Leu Ile Lys

65 70 75 80

Gln Glu Leu Asp Lys Tyr Lys Asn Ala Val Thr Glu Leu Gln Leu Leu

85 90 95

Met Gln Ser Thr Pro Ala Thr Asn Asn Arg Ala Arg Arg Glu Leu Pro

100 105 110

Arg Phe Met Asn Tyr Thr Leu Asn Asn Ala Lys Lys Thr Asn Val Thr

115 120 125

Leu Ser Lys Lys Arg Lys Asn Asn Phe Leu Gly Phe Cys Leu Gly Val

130 135 140

Gly Ser Ala Ile Ala Ser Gly Val Ala Val Ser Lys Val Leu His Leu

145 150 155 160

Glu Gly Glu Val Asn Lys Ile Lys Ser Ala Leu Leu Ser Thr Asn Lys

165 170 175

Ala Val Val Ser Leu Ser Asn Gly Val Ser Val Leu Val Val Lys Val

180 185 190

Leu Asp Leu Lys Asn Tyr Ile Asp Lys Gln Leu Leu Pro Ile Val Asn

195 200 205

Lys Gln Ser Cys Ser Ile Ser Asn Ile Glu Thr Val Ile Glu Phe Gln

210 215 220

Gln Lys Asn Asn Arg Leu Leu Glu Ile Thr Arg Glu Phe Ser Val Asn

225 230 235 240

Ala Gly Val Thr Thr Pro Val Ser Thr Tyr Met Leu Thr Asn Ser Glu

245 250 255

Leu Leu Ser Leu Ile Asn Asp Met Pro Ile Thr Asn Asp Gln Lys Lys

260 265 270

Leu Met Ser Asn Asn Val Gln Ile Val Arg Gln Gln Ser Tyr Ser Ile

275 280 285

Met Ser Ile Ile Lys Glu Glu Val Leu Ala Tyr Val Val Gln Leu Pro

290 295 300

Leu Tyr Gly Val Ile Asp Thr Pro Cys Trp Lys Leu His Thr Ser Pro

305 310 315 320

Leu Cys Thr Thr Asn Thr Lys Glu Gly Ser Asn Ile Cys Leu Thr Arg

325 330 335

Thr Asp Arg Gly Trp Tyr Cys Asp Asn Ala Gly Ser Val Ser Phe Phe

340 345 350

Pro Gln Ala Glu Thr Cys Lys Val Gln Ser Asn Arg Val Phe Cys Asp

355 360 365

Thr Met Asn Ser Cys Thr Leu Pro Ser Glu Val Asn Leu Cys Asn Thr

370 375 380

Asp Ile Phe Asn Pro Lys Tyr Asp Cys Lys Ile Met Thr Ser Lys Thr

385 390 395 400

Asp Val Ser Ser Ser Val Ile Thr Ser Leu Gly Ala Ile Val Ser Cys

405 410 415

Tyr Gly Lys Thr Lys Cys Thr Ala Ser Asn Lys Asn Arg Gly Ile Ile

420 425 430

Lys Thr Phe Ser Asn Gly Cys Asp Tyr Val Ser Asn Lys Gly Val Asp

435 440 445

Thr Val Ser Val Gly Asn Thr Leu Tyr Tyr Val Asn Lys Gln Glu Gly

450 455 460

Lys Ser Leu Tyr Val Lys Gly Glu Pro Ile Ile Asn Phe Tyr Asp Pro

465 470 475 480

Leu Val Phe Pro Ser Asp Glu Phe Asp Ala Ser Ile Ser Gln Val Asn

485 490 495

Glu Lys Ile Asn Gln Ser Leu Ala Phe Ile Arg Lys Ser Asp Glu Leu

500 505 510

Leu Gly Ser Gly Tyr Ile Pro Glu Ala Pro Arg Asp Gly Gln Ala Tyr

515 520 525

Val Arg Lys Asp Gly Glu Trp Val Leu Leu Ser Thr Phe Leu Asp Lys

530 535 540

Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro

545 550 555 560

Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser

565 570 575

Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp

580 585 590

Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn

595 600 605

Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val

610 615 620

Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu

625 630 635 640

Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys

645 650 655

Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr

660 665 670

Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr

675 680 685

Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu

690 695 700

Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu

705 710 715 720

Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys

725 730 735

Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu

740 745 750

Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

755 760 765

Lys

<210> 33

<211> 769

<212>PRT

<213> Artificial sequence

<220>

<223> FH_85-Fc

<400> 33

Met Glu Leu Leu Ile Leu Lys Ala Asn Ala Ile Thr Thr Ile Leu Thr

1 5 10 15

Ala Val Thr Phe Cys Phe Ala Ser Gly Gln Asn Ile Thr Glu Glu Phe

20 25 30

Tyr Gln Ser Thr Cys Ser Ala Val Ser Arg Gly Tyr Leu Ser Ala Leu

35 40 45

Arg Thr Gly Trp Tyr Thr Ser Val Ile Thr Ile Met Leu Ser Asn Ile

50 55 60

Lys Glu Asn Lys Cys Asn Gly Thr Asp Ala Lys Val Lys Leu Ile Lys

65 70 75 80

Gln Glu Leu Asp Lys Tyr Lys Asn Ala Val Thr Glu Leu Gln Leu Leu

85 90 95

Met Gln Ser Thr Pro Ala Thr Asn Asn Arg Ala Arg Arg Glu Leu Pro

100 105 110

Arg Phe Met Asn Tyr Thr Leu Asn Asn Ala Lys Lys Thr Asn Val Thr

115 120 125

Leu Ser Lys Lys Arg Lys Asn Asn Phe Leu Gly Phe Cys Leu Gly Val

130 135 140

Gly Ser Ala Ile Ala Ser Gly Val Ala Val Ser Lys Val Leu His Leu

145 150 155 160

Glu Gly Glu Val Asn Lys Ile Lys Ser Ala Leu Leu Ser Thr Asn Lys

165 170 175

Ala Val Val Ser Leu Ser Asn Gly Val Ser Val Leu Val Val Lys Val

180 185 190

Leu Asp Leu Lys Asn Tyr Ile Asp Lys Gln Leu Leu Pro Ile Val Asn

195 200 205

Lys Gln Ser Cys Ser Ile Ser Asn Ile Glu Thr Val Ile Glu Phe Gln

210 215 220

Gln Lys Asn Asn Arg Leu Leu Glu Ile Thr Arg Glu Phe Ser Val Asn

225 230 235 240

Ala Gly Val Thr Thr Pro Val Ser Thr Tyr Met Leu Thr Asn Ser Glu

245 250 255

Leu Leu Ser Leu Ile Asn Asp Met Pro Ile Thr Asn Asp Gln Lys Lys

260 265 270

Leu Met Ser Asn Asn Val Gln Ile Val Arg Gln Gln Ser Tyr Ser Ile

275 280 285

Met Ser Ile Ile Lys Glu Glu Val Leu Ala Tyr Val Val Gln Leu Pro

290 295 300

Leu Tyr Gly Val Ile Asp Thr Pro Cys Trp Lys Leu His Thr Ser Pro

305 310 315 320

Leu Cys Thr Thr Asn Thr Lys Glu Gly Ser Asn Ile Cys Leu Thr Arg

325 330 335

Thr Asp Arg Gly Trp Tyr Cys Asp Asn Ala Gly Ser Val Ser Phe Phe

340 345 350

Pro Gln Ala Glu Thr Cys Lys Val Gln Ser Asn Arg Val Phe Cys Asp

355 360 365

Thr Met Asn Ser Cys Thr Leu Pro Ser Glu Val Asn Leu Cys Asn Thr

370 375 380

Asp Ile Phe Asn Pro Lys Tyr Asp Cys Lys Ile Met Thr Ser Lys Thr

385 390 395 400

Asp Val Ser Ser Ser Val Ile Thr Ser Leu Gly Ala Ile Val Ser Cys

405 410 415

Tyr Gly Lys Thr Lys Cys Thr Ala Ser Asn Lys Asn Arg Gly Ile Ile

420 425 430

Lys Thr Phe Ser Asn Gly Cys Asp Tyr Val Ser Asn Lys Gly Val Asp

435 440 445

Thr Val Ser Val Gly Asn Thr Leu Tyr Tyr Val Asn Lys Gln Glu Gly

450 455 460

Lys Ser Leu Tyr Val Lys Gly Glu Pro Ile Ile Asn Phe Tyr Asp Pro

465 470 475 480

Leu Val Phe Pro Ser Asp Glu Phe Asp Ala Ser Ile Ser Gln Val Asn

485 490 495

Glu Lys Ile Asn Gln Ser Leu Ala Phe Ile Arg Lys Ser Asp Glu Leu

500 505 510

Leu Gly Ser Gly Tyr Ile Pro Glu Ala Pro Arg Asp Gly Gln Ala Tyr

515 520 525

Val Arg Lys Asp Gly Glu Trp Val Leu Leu Ser Thr Phe Leu Asp Lys

530 535 540

Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro

545 550 555 560

Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser

565 570 575

Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp

580 585 590

Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn

595 600 605

Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val

610 615 620

Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu

625 630 635 640

Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys

645 650 655

Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr

660 665 670

Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr

675 680 685

Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu

690 695 700

Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu

705 710 715 720

Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys

725 730 735

Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu

740 745 750

Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

755 760 765

Lys

<210> 34

<211> 789

<212>PRT

<213> Artificial sequence

<220>

<223> F_DS-Cav1-4GS-Fc

<400> 34

Met Glu Leu Leu Ile Leu Lys Ala Asn Ala Ile Thr Thr Ile Leu Thr

1 5 10 15

Ala Val Thr Phe Cys Phe Ala Ser Gly Gln Asn Ile Thr Glu Glu Phe

20 25 30

Tyr Gln Ser Thr Cys Ser Ala Val Ser Lys Gly Tyr Leu Ser Ala Leu

35 40 45

Arg Thr Gly Trp Tyr Thr Ser Val Ile Thr Ile Glu Leu Ser Asn Ile

50 55 60

Lys Glu Asn Lys Cys Asn Gly Thr Asp Ala Lys Val Lys Leu Ile Lys

65 70 75 80

Gln Glu Leu Asp Lys Tyr Lys Asn Ala Val Thr Glu Leu Gln Leu Leu

85 90 95

Met Gln Ser Thr Pro Ala Thr Asn Asn Arg Ala Arg Arg Glu Leu Pro

100 105 110

Arg Phe Met Asn Tyr Thr Leu Asn Asn Ala Lys Lys Thr Asn Val Thr

115 120 125

Leu Ser Lys Lys Arg Lys Arg Arg Phe Leu Gly Phe Leu Leu Gly Val

130 135 140

Gly Ser Ala Ile Ala Ser Gly Val Ala Val Cys Lys Val Leu His Leu

145 150 155 160

Glu Gly Glu Val Asn Lys Ile Lys Ser Ala Leu Leu Ser Thr Asn Lys

165 170 175

Ala Val Val Ser Leu Ser Asn Gly Val Ser Val Leu Thr Phe Lys Val

180 185 190

Leu Asp Leu Lys Asn Tyr Ile Asp Lys Gln Leu Leu Pro Ile Leu Asn

195 200 205

Lys Gln Ser Cys Ser Ile Ser Asn Ile Glu Thr Val Ile Glu Phe Gln

210 215 220

Gln Lys Asn Asn Arg Leu Leu Glu Ile Thr Arg Glu Phe Ser Val Asn

225 230 235 240

Ala Gly Val Thr Thr Pro Val Ser Thr Tyr Met Leu Thr Asn Ser Glu

245 250 255

Leu Leu Ser Leu Ile Asn Asp Met Pro Ile Thr Asn Asp Gln Lys Lys

260 265 270

Leu Met Ser Asn Asn Val Gln Ile Val Arg Gln Gln Ser Tyr Ser Ile

275 280 285

Met Cys Ile Ile Lys Glu Glu Val Leu Ala Tyr Val Val Gln Leu Pro

290 295 300

Leu Tyr Gly Val Ile Asp Thr Pro Cys Trp Lys Leu His Thr Ser Pro

305 310 315 320

Leu Cys Thr Thr Asn Thr Lys Glu Gly Ser Asn Ile Cys Leu Thr Arg

325 330 335

Thr Asp Arg Gly Trp Tyr Cys Asp Asn Ala Gly Ser Val Ser Phe Phe

340 345 350

Pro Gln Ala Glu Thr Cys Lys Val Gln Ser Asn Arg Val Phe Cys Asp

355 360 365

Thr Met Asn Ser Leu Thr Leu Pro Ser Glu Val Asn Leu Cys Asn Val

370 375 380

Asp Ile Phe Asn Pro Lys Tyr Asp Cys Lys Ile Met Thr Ser Lys Thr

385 390 395 400

Asp Val Ser Ser Ser Val Ile Thr Ser Leu Gly Ala Ile Val Ser Cys

405 410 415

Tyr Gly Lys Thr Lys Cys Thr Ala Ser Asn Lys Asn Arg Gly Ile Ile

420 425 430

Lys Thr Phe Ser Asn Gly Cys Asp Tyr Val Ser Asn Lys Gly Val Asp

435 440 445

Thr Val Ser Val Gly Asn Thr Leu Tyr Tyr Val Asn Lys Gln Glu Gly

450 455 460

Lys Ser Leu Tyr Val Lys Gly Glu Pro Ile Ile Asn Phe Tyr Asp Pro

465 470 475 480

Leu Val Phe Pro Ser Asp Glu Phe Asp Ala Ser Ile Ser Gln Val Asn

485 490 495

Glu Lys Ile Asn Gln Ser Leu Ala Phe Ile Arg Lys Ser Asp Glu Leu

500 505 510

Leu Gly Ser Gly Tyr Ile Pro Glu Ala Pro Arg Asp Gly Gln Ala Tyr

515 520 525

Val Arg Lys Asp Gly Glu Trp Val Leu Leu Ser Thr Phe Leu Gly Gly

530 535 540

Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly

545 550 555 560

Gly Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu

565 570 575

Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr

580 585 590

Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val

595 600 605

Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val

610 615 620

Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser

625 630 635 640

Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu

645 650 655

Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala

660 665 670

Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro

675 680 685

Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln

690 695 700

Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala

705 710 715 720

Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr

725 730 735

Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu

740 745 750

Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser

755 760 765

Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser

770 775 780

Leu Ser Pro Gly Lys

785

<210> 35

<211> 779

<212>PRT

<213> Artificial sequence

<220>

<223> F_DS-Cav1-2GS-Fc

<400> 35

Met Glu Leu Leu Ile Leu Lys Ala Asn Ala Ile Thr Thr Ile Leu Thr

1 5 10 15

Ala Val Thr Phe Cys Phe Ala Ser Gly Gln Asn Ile Thr Glu Glu Phe

20 25 30

Tyr Gln Ser Thr Cys Ser Ala Val Ser Lys Gly Tyr Leu Ser Ala Leu

35 40 45

Arg Thr Gly Trp Tyr Thr Ser Val Ile Thr Ile Glu Leu Ser Asn Ile

50 55 60

Lys Glu Asn Lys Cys Asn Gly Thr Asp Ala Lys Val Lys Leu Ile Lys

65 70 75 80

Gln Glu Leu Asp Lys Tyr Lys Asn Ala Val Thr Glu Leu Gln Leu Leu

85 90 95

Met Gln Ser Thr Pro Ala Thr Asn Asn Arg Ala Arg Arg Glu Leu Pro

100 105 110

Arg Phe Met Asn Tyr Thr Leu Asn Asn Ala Lys Lys Thr Asn Val Thr

115 120 125

Leu Ser Lys Lys Arg Lys Arg Arg Phe Leu Gly Phe Leu Leu Gly Val

130 135 140

Gly Ser Ala Ile Ala Ser Gly Val Ala Val Cys Lys Val Leu His Leu

145 150 155 160

Glu Gly Glu Val Asn Lys Ile Lys Ser Ala Leu Leu Ser Thr Asn Lys

165 170 175

Ala Val Val Ser Leu Ser Asn Gly Val Ser Val Leu Thr Phe Lys Val

180 185 190

Leu Asp Leu Lys Asn Tyr Ile Asp Lys Gln Leu Leu Pro Ile Leu Asn

195 200 205

Lys Gln Ser Cys Ser Ile Ser Asn Ile Glu Thr Val Ile Glu Phe Gln

210 215 220

Gln Lys Asn Asn Arg Leu Leu Glu Ile Thr Arg Glu Phe Ser Val Asn

225 230 235 240

Ala Gly Val Thr Thr Pro Val Ser Thr Tyr Met Leu Thr Asn Ser Glu

245 250 255

Leu Leu Ser Leu Ile Asn Asp Met Pro Ile Thr Asn Asp Gln Lys Lys

260 265 270

Leu Met Ser Asn Asn Val Gln Ile Val Arg Gln Gln Ser Tyr Ser Ile

275 280 285

Met Cys Ile Ile Lys Glu Glu Val Leu Ala Tyr Val Val Gln Leu Pro

290 295 300

Leu Tyr Gly Val Ile Asp Thr Pro Cys Trp Lys Leu His Thr Ser Pro

305 310 315 320

Leu Cys Thr Thr Asn Thr Lys Glu Gly Ser Asn Ile Cys Leu Thr Arg

325 330 335

Thr Asp Arg Gly Trp Tyr Cys Asp Asn Ala Gly Ser Val Ser Phe Phe

340 345 350

Pro Gln Ala Glu Thr Cys Lys Val Gln Ser Asn Arg Val Phe Cys Asp

355 360 365

Thr Met Asn Ser Leu Thr Leu Pro Ser Glu Val Asn Leu Cys Asn Val

370 375 380

Asp Ile Phe Asn Pro Lys Tyr Asp Cys Lys Ile Met Thr Ser Lys Thr

385 390 395 400

Asp Val Ser Ser Ser Val Ile Thr Ser Leu Gly Ala Ile Val Ser Cys

405 410 415

Tyr Gly Lys Thr Lys Cys Thr Ala Ser Asn Lys Asn Arg Gly Ile Ile

420 425 430

Lys Thr Phe Ser Asn Gly Cys Asp Tyr Val Ser Asn Lys Gly Val Asp

435 440 445

Thr Val Ser Val Gly Asn Thr Leu Tyr Tyr Val Asn Lys Gln Glu Gly

450 455 460

Lys Ser Leu Tyr Val Lys Gly Glu Pro Ile Ile Asn Phe Tyr Asp Pro

465 470 475 480

Leu Val Phe Pro Ser Asp Glu Phe Asp Ala Ser Ile Ser Gln Val Asn

485 490 495

Glu Lys Ile Asn Gln Ser Leu Ala Phe Ile Arg Lys Ser Asp Glu Leu

500 505 510

Leu Gly Ser Gly Tyr Ile Pro Glu Ala Pro Arg Asp Gly Gln Ala Tyr

515 520 525

Val Arg Lys Asp Gly Glu Trp Val Leu Leu Ser Thr Phe Leu Gly Gly

530 535 540

Gly Gly Ser Gly Gly Gly Gly Ser Asp Lys Thr His Thr Cys Pro Pro

545 550 555 560

Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro

565 570 575

Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr

580 585 590

Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn

595 600 605

Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg

610 615 620

Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val

625 630 635 640

Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser

645 650 655

Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys

660 665 670

Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp

675 680 685

Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe

690 695 700

Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu

705 710 715 720

Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe

725 730 735

Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly

740 745 750

Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr

755 760 765

Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

770 775

<210> 36

<211> 774

<212>PRT

<213> Artificial sequence

<220>

<223> F_DS-Cav1-1GS-Fc

<400> 36

Met Glu Leu Leu Ile Leu Lys Ala Asn Ala Ile Thr Thr Ile Leu Thr

1 5 10 15

Ala Val Thr Phe Cys Phe Ala Ser Gly Gln Asn Ile Thr Glu Glu Phe

20 25 30

Tyr Gln Ser Thr Cys Ser Ala Val Ser Lys Gly Tyr Leu Ser Ala Leu

35 40 45

Arg Thr Gly Trp Tyr Thr Ser Val Ile Thr Ile Glu Leu Ser Asn Ile

50 55 60

Lys Glu Asn Lys Cys Asn Gly Thr Asp Ala Lys Val Lys Leu Ile Lys

65 70 75 80

Gln Glu Leu Asp Lys Tyr Lys Asn Ala Val Thr Glu Leu Gln Leu Leu

85 90 95

Met Gln Ser Thr Pro Ala Thr Asn Asn Arg Ala Arg Arg Glu Leu Pro

100 105 110

Arg Phe Met Asn Tyr Thr Leu Asn Asn Ala Lys Lys Thr Asn Val Thr

115 120 125

Leu Ser Lys Lys Arg Lys Arg Arg Phe Leu Gly Phe Leu Leu Gly Val

130 135 140

Gly Ser Ala Ile Ala Ser Gly Val Ala Val Cys Lys Val Leu His Leu

145 150 155 160

Glu Gly Glu Val Asn Lys Ile Lys Ser Ala Leu Leu Ser Thr Asn Lys

165 170 175

Ala Val Val Ser Leu Ser Asn Gly Val Ser Val Leu Thr Phe Lys Val

180 185 190

Leu Asp Leu Lys Asn Tyr Ile Asp Lys Gln Leu Leu Pro Ile Leu Asn

195 200 205

Lys Gln Ser Cys Ser Ile Ser Asn Ile Glu Thr Val Ile Glu Phe Gln

210 215 220

Gln Lys Asn Asn Arg Leu Leu Glu Ile Thr Arg Glu Phe Ser Val Asn

225 230 235 240

Ala Gly Val Thr Thr Pro Val Ser Thr Tyr Met Leu Thr Asn Ser Glu

245 250 255

Leu Leu Ser Leu Ile Asn Asp Met Pro Ile Thr Asn Asp Gln Lys Lys

260 265 270

Leu Met Ser Asn Asn Val Gln Ile Val Arg Gln Gln Ser Tyr Ser Ile

275 280 285

Met Cys Ile Ile Lys Glu Glu Val Leu Ala Tyr Val Val Gln Leu Pro

290 295 300

Leu Tyr Gly Val Ile Asp Thr Pro Cys Trp Lys Leu His Thr Ser Pro

305 310 315 320

Leu Cys Thr Thr Asn Thr Lys Glu Gly Ser Asn Ile Cys Leu Thr Arg

325 330 335

Thr Asp Arg Gly Trp Tyr Cys Asp Asn Ala Gly Ser Val Ser Phe Phe

340 345 350

Pro Gln Ala Glu Thr Cys Lys Val Gln Ser Asn Arg Val Phe Cys Asp

355 360 365

Thr Met Asn Ser Leu Thr Leu Pro Ser Glu Val Asn Leu Cys Asn Val

370 375 380

Asp Ile Phe Asn Pro Lys Tyr Asp Cys Lys Ile Met Thr Ser Lys Thr

385 390 395 400

Asp Val Ser Ser Ser Val Ile Thr Ser Leu Gly Ala Ile Val Ser Cys

405 410 415

Tyr Gly Lys Thr Lys Cys Thr Ala Ser Asn Lys Asn Arg Gly Ile Ile

420 425 430

Lys Thr Phe Ser Asn Gly Cys Asp Tyr Val Ser Asn Lys Gly Val Asp

435 440 445

Thr Val Ser Val Gly Asn Thr Leu Tyr Tyr Val Asn Lys Gln Glu Gly

450 455 460

Lys Ser Leu Tyr Val Lys Gly Glu Pro Ile Ile Asn Phe Tyr Asp Pro

465 470 475 480

Leu Val Phe Pro Ser Asp Glu Phe Asp Ala Ser Ile Ser Gln Val Asn

485 490 495

Glu Lys Ile Asn Gln Ser Leu Ala Phe Ile Arg Lys Ser Asp Glu Leu

500 505 510

Leu Gly Ser Gly Tyr Ile Pro Glu Ala Pro Arg Asp Gly Gln Ala Tyr

515 520 525

Val Arg Lys Asp Gly Glu Trp Val Leu Leu Ser Thr Phe Leu Gly Gly

530 535 540

Gly Gly Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu

545 550 555 560

Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp

565 570 575

Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp

580 585 590

Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly

595 600 605

Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn

610 615 620

Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp

625 630 635 640

Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro

645 650 655

Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu

660 665 670

Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn

675 680 685

Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile

690 695 700

Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr

705 710 715 720

Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys

725 730 735

Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys

740 745 750

Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu

755 760 765

Ser Leu Ser Pro Gly Lys

770

<210> 37

<211> 789

<212>PRT

<213> Artificial sequence

<220>

<223> FH_85-4GS-Fc

<400> 37

Met Glu Leu Leu Ile Leu Lys Ala Asn Ala Ile Thr Thr Ile Leu Thr

1 5 10 15

Ala Val Thr Phe Cys Phe Ala Ser Gly Gln Asn Ile Thr Glu Glu Phe

20 25 30

Tyr Gln Ser Thr Cys Ser Ala Val Ser Arg Gly Tyr Leu Ser Ala Leu

35 40 45

Arg Thr Gly Trp Tyr Thr Ser Val Ile Thr Ile Met Leu Ser Asn Ile

50 55 60

Lys Glu Asn Lys Cys Asn Gly Thr Asp Ala Lys Val Lys Leu Ile Lys

65 70 75 80

Gln Glu Leu Asp Lys Tyr Lys Asn Ala Val Thr Glu Leu Gln Leu Leu

85 90 95

Met Gln Ser Thr Pro Ala Thr Asn Asn Arg Ala Arg Arg Glu Leu Pro

100 105 110

Arg Phe Met Asn Tyr Thr Leu Asn Asn Ala Lys Lys Thr Asn Val Thr

115 120 125

Leu Ser Lys Lys Arg Lys Asn Asn Phe Leu Gly Phe Cys Leu Gly Val

130 135 140

Gly Ser Ala Ile Ala Ser Gly Val Ala Val Ser Lys Val Leu His Leu

145 150 155 160

Glu Gly Glu Val Asn Lys Ile Lys Ser Ala Leu Leu Ser Thr Asn Lys

165 170 175

Ala Val Val Ser Leu Ser Asn Gly Val Ser Val Leu Val Val Lys Val

180 185 190

Leu Asp Leu Lys Asn Tyr Ile Asp Lys Gln Leu Leu Pro Ile Val Asn

195 200 205

Lys Gln Ser Cys Ser Ile Ser Asn Ile Glu Thr Val Ile Glu Phe Gln

210 215 220

Gln Lys Asn Asn Arg Leu Leu Glu Ile Thr Arg Glu Phe Ser Val Asn

225 230 235 240

Ala Gly Val Thr Thr Pro Val Ser Thr Tyr Met Leu Thr Asn Ser Glu

245 250 255

Leu Leu Ser Leu Ile Asn Asp Met Pro Ile Thr Asn Asp Gln Lys Lys

260 265 270

Leu Met Ser Asn Asn Val Gln Ile Val Arg Gln Gln Ser Tyr Ser Ile

275 280 285

Met Ser Ile Ile Lys Glu Glu Val Leu Ala Tyr Val Val Gln Leu Pro

290 295 300

Leu Tyr Gly Val Ile Asp Thr Pro Cys Trp Lys Leu His Thr Ser Pro

305 310 315 320

Leu Cys Thr Thr Asn Thr Lys Glu Gly Ser Asn Ile Cys Leu Thr Arg

325 330 335

Thr Asp Arg Gly Trp Tyr Cys Asp Asn Ala Gly Ser Val Ser Phe Phe

340 345 350

Pro Gln Ala Glu Thr Cys Lys Val Gln Ser Asn Arg Val Phe Cys Asp

355 360 365

Thr Met Asn Ser Cys Thr Leu Pro Ser Glu Val Asn Leu Cys Asn Thr

370 375 380

Asp Ile Phe Asn Pro Lys Tyr Asp Cys Lys Ile Met Thr Ser Lys Thr

385 390 395 400

Asp Val Ser Ser Ser Val Ile Thr Ser Leu Gly Ala Ile Val Ser Cys

405 410 415

Tyr Gly Lys Thr Lys Cys Thr Ala Ser Asn Lys Asn Arg Gly Ile Ile

420 425 430

Lys Thr Phe Ser Asn Gly Cys Asp Tyr Val Ser Asn Lys Gly Val Asp

435 440 445

Thr Val Ser Val Gly Asn Thr Leu Tyr Tyr Val Asn Lys Gln Glu Gly

450 455 460

Lys Ser Leu Tyr Val Lys Gly Glu Pro Ile Ile Asn Phe Tyr Asp Pro

465 470 475 480

Leu Val Phe Pro Ser Asp Glu Phe Asp Ala Ser Ile Ser Gln Val Asn

485 490 495

Glu Lys Ile Asn Gln Ser Leu Ala Phe Ile Arg Lys Ser Asp Glu Leu

500 505 510

Leu Gly Ser Gly Tyr Ile Pro Glu Ala Pro Arg Asp Gly Gln Ala Tyr

515 520 525

Val Arg Lys Asp Gly Glu Trp Val Leu Leu Ser Thr Phe Leu Gly Gly

530 535 540

Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly

545 550 555 560

Gly Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu

565 570 575

Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr

580 585 590

Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val

595 600 605

Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val

610 615 620

Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser

625 630 635 640

Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu

645 650 655

Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala

660 665 670

Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro

675 680 685

Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln

690 695 700

Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala

705 710 715 720

Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr

725 730 735

Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu

740 745 750

Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser

755 760 765

Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser

770 775 780

Leu Ser Pro Gly Lys

785

<210> 38

<211> 779

<212>PRT

<213> Artificial sequence

<220>

<223> FH_85-2GS-Fc

<400> 38

Met Glu Leu Leu Ile Leu Lys Ala Asn Ala Ile Thr Thr Ile Leu Thr

1 5 10 15

Ala Val Thr Phe Cys Phe Ala Ser Gly Gln Asn Ile Thr Glu Glu Phe

20 25 30

Tyr Gln Ser Thr Cys Ser Ala Val Ser Arg Gly Tyr Leu Ser Ala Leu

35 40 45

Arg Thr Gly Trp Tyr Thr Ser Val Ile Thr Ile Met Leu Ser Asn Ile

50 55 60

Lys Glu Asn Lys Cys Asn Gly Thr Asp Ala Lys Val Lys Leu Ile Lys

65 70 75 80

Gln Glu Leu Asp Lys Tyr Lys Asn Ala Val Thr Glu Leu Gln Leu Leu

85 90 95

Met Gln Ser Thr Pro Ala Thr Asn Asn Arg Ala Arg Arg Glu Leu Pro

100 105 110

Arg Phe Met Asn Tyr Thr Leu Asn Asn Ala Lys Lys Thr Asn Val Thr

115 120 125

Leu Ser Lys Lys Arg Lys Asn Asn Phe Leu Gly Phe Cys Leu Gly Val

130 135 140

Gly Ser Ala Ile Ala Ser Gly Val Ala Val Ser Lys Val Leu His Leu

145 150 155 160

Glu Gly Glu Val Asn Lys Ile Lys Ser Ala Leu Leu Ser Thr Asn Lys

165 170 175

Ala Val Val Ser Leu Ser Asn Gly Val Ser Val Leu Val Val Lys Val

180 185 190

Leu Asp Leu Lys Asn Tyr Ile Asp Lys Gln Leu Leu Pro Ile Val Asn

195 200 205

Lys Gln Ser Cys Ser Ile Ser Asn Ile Glu Thr Val Ile Glu Phe Gln

210 215 220

Gln Lys Asn Asn Arg Leu Leu Glu Ile Thr Arg Glu Phe Ser Val Asn

225 230 235 240

Ala Gly Val Thr Thr Pro Val Ser Thr Tyr Met Leu Thr Asn Ser Glu

245 250 255

Leu Leu Ser Leu Ile Asn Asp Met Pro Ile Thr Asn Asp Gln Lys Lys

260 265 270

Leu Met Ser Asn Asn Val Gln Ile Val Arg Gln Gln Ser Tyr Ser Ile

275 280 285

Met Ser Ile Ile Lys Glu Glu Val Leu Ala Tyr Val Val Gln Leu Pro

290 295 300

Leu Tyr Gly Val Ile Asp Thr Pro Cys Trp Lys Leu His Thr Ser Pro

305 310 315 320

Leu Cys Thr Thr Asn Thr Lys Glu Gly Ser Asn Ile Cys Leu Thr Arg

325 330 335

Thr Asp Arg Gly Trp Tyr Cys Asp Asn Ala Gly Ser Val Ser Phe Phe

340 345 350

Pro Gln Ala Glu Thr Cys Lys Val Gln Ser Asn Arg Val Phe Cys Asp

355 360 365

Thr Met Asn Ser Cys Thr Leu Pro Ser Glu Val Asn Leu Cys Asn Thr

370 375 380

Asp Ile Phe Asn Pro Lys Tyr Asp Cys Lys Ile Met Thr Ser Lys Thr

385 390 395 400

Asp Val Ser Ser Ser Val Ile Thr Ser Leu Gly Ala Ile Val Ser Cys

405 410 415

Tyr Gly Lys Thr Lys Cys Thr Ala Ser Asn Lys Asn Arg Gly Ile Ile

420 425 430

Lys Thr Phe Ser Asn Gly Cys Asp Tyr Val Ser Asn Lys Gly Val Asp

435 440 445

Thr Val Ser Val Gly Asn Thr Leu Tyr Tyr Val Asn Lys Gln Glu Gly

450 455 460

Lys Ser Leu Tyr Val Lys Gly Glu Pro Ile Ile Asn Phe Tyr Asp Pro

465 470 475 480

Leu Val Phe Pro Ser Asp Glu Phe Asp Ala Ser Ile Ser Gln Val Asn

485 490 495

Glu Lys Ile Asn Gln Ser Leu Ala Phe Ile Arg Lys Ser Asp Glu Leu

500 505 510

Leu Gly Ser Gly Tyr Ile Pro Glu Ala Pro Arg Asp Gly Gln Ala Tyr

515 520 525

Val Arg Lys Asp Gly Glu Trp Val Leu Leu Ser Thr Phe Leu Gly Gly

530 535 540

Gly Gly Ser Gly Gly Gly Gly Ser Asp Lys Thr His Thr Cys Pro Pro

545 550 555 560

Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro

565 570 575

Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr

580 585 590

Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn

595 600 605

Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg

610 615 620

Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val

625 630 635 640

Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser

645 650 655

Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys

660 665 670

Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp

675 680 685

Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe

690 695 700

Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu

705 710 715 720

Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe

725 730 735

Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly

740 745 750

Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr

755 760 765

Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

770 775

<210> 39

<211> 774

<212>PRT

<213> Artificial sequence

<220>

<223> FH_85-1GS-Fc

<400> 39

Met Glu Leu Leu Ile Leu Lys Ala Asn Ala Ile Thr Thr Ile Leu Thr

1 5 10 15

Ala Val Thr Phe Cys Phe Ala Ser Gly Gln Asn Ile Thr Glu Glu Phe

20 25 30

Tyr Gln Ser Thr Cys Ser Ala Val Ser Arg Gly Tyr Leu Ser Ala Leu

35 40 45

Arg Thr Gly Trp Tyr Thr Ser Val Ile Thr Ile Met Leu Ser Asn Ile

50 55 60

Lys Glu Asn Lys Cys Asn Gly Thr Asp Ala Lys Val Lys Leu Ile Lys

65 70 75 80

Gln Glu Leu Asp Lys Tyr Lys Asn Ala Val Thr Glu Leu Gln Leu Leu

85 90 95

Met Gln Ser Thr Pro Ala Thr Asn Asn Arg Ala Arg Arg Glu Leu Pro

100 105 110

Arg Phe Met Asn Tyr Thr Leu Asn Asn Ala Lys Lys Thr Asn Val Thr

115 120 125

Leu Ser Lys Lys Arg Lys Asn Asn Phe Leu Gly Phe Cys Leu Gly Val

130 135 140

Gly Ser Ala Ile Ala Ser Gly Val Ala Val Ser Lys Val Leu His Leu

145 150 155 160

Glu Gly Glu Val Asn Lys Ile Lys Ser Ala Leu Leu Ser Thr Asn Lys

165 170 175

Ala Val Val Ser Leu Ser Asn Gly Val Ser Val Leu Val Val Lys Val

180 185 190

Leu Asp Leu Lys Asn Tyr Ile Asp Lys Gln Leu Leu Pro Ile Val Asn

195 200 205

Lys Gln Ser Cys Ser Ile Ser Asn Ile Glu Thr Val Ile Glu Phe Gln

210 215 220

Gln Lys Asn Asn Arg Leu Leu Glu Ile Thr Arg Glu Phe Ser Val Asn

225 230 235 240

Ala Gly Val Thr Thr Pro Val Ser Thr Tyr Met Leu Thr Asn Ser Glu

245 250 255

Leu Leu Ser Leu Ile Asn Asp Met Pro Ile Thr Asn Asp Gln Lys Lys

260 265 270

Leu Met Ser Asn Asn Val Gln Ile Val Arg Gln Gln Ser Tyr Ser Ile

275 280 285

Met Ser Ile Ile Lys Glu Glu Val Leu Ala Tyr Val Val Gln Leu Pro

290 295 300

Leu Tyr Gly Val Ile Asp Thr Pro Cys Trp Lys Leu His Thr Ser Pro

305 310 315 320

Leu Cys Thr Thr Asn Thr Lys Glu Gly Ser Asn Ile Cys Leu Thr Arg

325 330 335

Thr Asp Arg Gly Trp Tyr Cys Asp Asn Ala Gly Ser Val Ser Phe Phe

340 345 350

Pro Gln Ala Glu Thr Cys Lys Val Gln Ser Asn Arg Val Phe Cys Asp

355 360 365

Thr Met Asn Ser Cys Thr Leu Pro Ser Glu Val Asn Leu Cys Asn Thr

370 375 380

Asp Ile Phe Asn Pro Lys Tyr Asp Cys Lys Ile Met Thr Ser Lys Thr

385 390 395 400

Asp Val Ser Ser Ser Val Ile Thr Ser Leu Gly Ala Ile Val Ser Cys

405 410 415

Tyr Gly Lys Thr Lys Cys Thr Ala Ser Asn Lys Asn Arg Gly Ile Ile

420 425 430

Lys Thr Phe Ser Asn Gly Cys Asp Tyr Val Ser Asn Lys Gly Val Asp

435 440 445

Thr Val Ser Val Gly Asn Thr Leu Tyr Tyr Val Asn Lys Gln Glu Gly

450 455 460

Lys Ser Leu Tyr Val Lys Gly Glu Pro Ile Ile Asn Phe Tyr Asp Pro

465 470 475 480

Leu Val Phe Pro Ser Asp Glu Phe Asp Ala Ser Ile Ser Gln Val Asn

485 490 495

Glu Lys Ile Asn Gln Ser Leu Ala Phe Ile Arg Lys Ser Asp Glu Leu

500 505 510

Leu Gly Ser Gly Tyr Ile Pro Glu Ala Pro Arg Asp Gly Gln Ala Tyr

515 520 525

Val Arg Lys Asp Gly Glu Trp Val Leu Leu Ser Thr Phe Leu Gly Gly

530 535 540

Gly Gly Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu

545 550 555 560

Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp

565 570 575

Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp

580 585 590

Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly

595 600 605

Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn

610 615 620

Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp

625 630 635 640

Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro

645 650 655

Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu

660 665 670

Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn

675 680 685

Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile

690 695 700

Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr

705 710 715 720

Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys

725 730 735

Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys

740 745 750

Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu

755 760 765

Ser Leu Ser Pro Gly Lys

770

<210> 40

<211> 21

<212>PRT

<213> Artificial sequence

<220>

<223> CC02

<400> 40

Ile Ala Leu Ala Leu Glu Lys Ile Ala Leu Ala Leu Glu Lys Ile Ala

1 5 10 15

Leu Ala Leu Glu Lys

20

<210> 41

<211> 21

<212>PRT

<213> Artificial sequence

<220>

<223>CC03

<400> 41

Ile Lys Leu Glu Leu Glu Lys Ile Lys Leu Glu Leu Glu Lys Ile Lys

1 5 10 15

Leu Glu Leu Glu Lys

20

<210> 42

<211> 21

<212>PRT

<213> Artificial sequence

<220>

<223> CC07

<400> 42

Leu His Leu His Ile Glu Lys Leu His Leu His Ile Glu Lys Leu His

1 5 10 15

Leu His Ile Glu Lys

20

<210> 43

<211> 21

<212>PRT

<213> Artificial sequence

<220>

<223>CCO08

<400> 43

Leu Lys Leu Glu Ile His His Leu Lys Leu Glu Ile His His Leu Lys

1 5 10 15

Leu Glu Ile His His

20

<210> 44

<211> 571

<212>PRT

<213> Artificial sequence

<220>

<223> F_DS-Cav1-CC02

<400> 44

Met Glu Leu Leu Ile Leu Lys Ala Asn Ala Ile Thr Thr Ile Leu Thr

1 5 10 15

Ala Val Thr Phe Cys Phe Ala Ser Gly Gln Asn Ile Thr Glu Glu Phe

20 25 30

Tyr Gln Ser Thr Cys Ser Ala Val Ser Lys Gly Tyr Leu Ser Ala Leu

35 40 45

Arg Thr Gly Trp Tyr Thr Ser Val Ile Thr Ile Glu Leu Ser Asn Ile

50 55 60

Lys Glu Asn Lys Cys Asn Gly Thr Asp Ala Lys Val Lys Leu Ile Lys

65 70 75 80

Gln Glu Leu Asp Lys Tyr Lys Asn Ala Val Thr Glu Leu Gln Leu Leu

85 90 95

Met Gln Ser Thr Pro Ala Thr Asn Asn Arg Ala Arg Arg Glu Leu Pro

100 105 110

Arg Phe Met Asn Tyr Thr Leu Asn Asn Ala Lys Lys Thr Asn Val Thr

115 120 125

Leu Ser Lys Lys Arg Lys Arg Arg Phe Leu Gly Phe Leu Leu Gly Val

130 135 140

Gly Ser Ala Ile Ala Ser Gly Val Ala Val Cys Lys Val Leu His Leu

145 150 155 160

Glu Gly Glu Val Asn Lys Ile Lys Ser Ala Leu Leu Ser Thr Asn Lys

165 170 175

Ala Val Val Ser Leu Ser Asn Gly Val Ser Val Leu Thr Phe Lys Val

180 185 190

Leu Asp Leu Lys Asn Tyr Ile Asp Lys Gln Leu Leu Pro Ile Leu Asn

195 200 205

Lys Gln Ser Cys Ser Ile Ser Asn Ile Glu Thr Val Ile Glu Phe Gln

210 215 220

Gln Lys Asn Asn Arg Leu Leu Glu Ile Thr Arg Glu Phe Ser Val Asn

225 230 235 240

Ala Gly Val Thr Thr Pro Val Ser Thr Tyr Met Leu Thr Asn Ser Glu

245 250 255

Leu Leu Ser Leu Ile Asn Asp Met Pro Ile Thr Asn Asp Gln Lys Lys

260 265 270

Leu Met Ser Asn Asn Val Gln Ile Val Arg Gln Gln Ser Tyr Ser Ile

275 280 285

Met Cys Ile Ile Lys Glu Glu Val Leu Ala Tyr Val Val Gln Leu Pro

290 295 300

Leu Tyr Gly Val Ile Asp Thr Pro Cys Trp Lys Leu His Thr Ser Pro

305 310 315 320

Leu Cys Thr Thr Asn Thr Lys Glu Gly Ser Asn Ile Cys Leu Thr Arg

325 330 335

Thr Asp Arg Gly Trp Tyr Cys Asp Asn Ala Gly Ser Val Ser Phe Phe

340 345 350

Pro Gln Ala Glu Thr Cys Lys Val Gln Ser Asn Arg Val Phe Cys Asp

355 360 365

Thr Met Asn Ser Leu Thr Leu Pro Ser Glu Val Asn Leu Cys Asn Val

370 375 380

Asp Ile Phe Asn Pro Lys Tyr Asp Cys Lys Ile Met Thr Ser Lys Thr

385 390 395 400

Asp Val Ser Ser Ser Val Ile Thr Ser Leu Gly Ala Ile Val Ser Cys

405 410 415

Tyr Gly Lys Thr Lys Cys Thr Ala Ser Asn Lys Asn Arg Gly Ile Ile

420 425 430

Lys Thr Phe Ser Asn Gly Cys Asp Tyr Val Ser Asn Lys Gly Val Asp

435 440 445

Thr Val Ser Val Gly Asn Thr Leu Tyr Tyr Val Asn Lys Gln Glu Gly

450 455 460

Lys Ser Leu Tyr Val Lys Gly Glu Pro Ile Ile Asn Phe Tyr Asp Pro

465 470 475 480

Leu Val Phe Pro Ser Asp Glu Phe Asp Ala Ser Ile Ser Gln Val Asn

485 490 495

Glu Lys Ile Asn Gln Ser Leu Ala Phe Ile Arg Lys Ser Asp Glu Leu

500 505 510

Leu Gly Ser Gly Tyr Ile Pro Glu Ala Pro Arg Asp Gly Gln Ala Tyr

515 520 525

Val Arg Lys Asp Gly Glu Trp Val Leu Leu Ser Thr Phe Leu Ile Ala

530 535 540

Leu Ala Leu Glu Lys Ile Ala Leu Ala Leu Glu Lys Ile Ala Leu Ala

545 550 555 560

Leu Glu Lys Asp Tyr Lys Asp Asp Asp Asp Lys

565 570

<210> 45

<211> 571

<212>PRT

<213> Artificial sequence

<220>

<223> F_DS-Cav1-CC03

<400> 45

Met Glu Leu Leu Ile Leu Lys Ala Asn Ala Ile Thr Thr Ile Leu Thr

1 5 10 15

Ala Val Thr Phe Cys Phe Ala Ser Gly Gln Asn Ile Thr Glu Glu Phe

20 25 30

Tyr Gln Ser Thr Cys Ser Ala Val Ser Lys Gly Tyr Leu Ser Ala Leu

35 40 45

Arg Thr Gly Trp Tyr Thr Ser Val Ile Thr Ile Glu Leu Ser Asn Ile

50 55 60

Lys Glu Asn Lys Cys Asn Gly Thr Asp Ala Lys Val Lys Leu Ile Lys

65 70 75 80

Gln Glu Leu Asp Lys Tyr Lys Asn Ala Val Thr Glu Leu Gln Leu Leu

85 90 95

Met Gln Ser Thr Pro Ala Thr Asn Asn Arg Ala Arg Arg Glu Leu Pro

100 105 110

Arg Phe Met Asn Tyr Thr Leu Asn Asn Ala Lys Lys Thr Asn Val Thr

115 120 125

Leu Ser Lys Lys Arg Lys Arg Arg Phe Leu Gly Phe Leu Leu Gly Val

130 135 140

Gly Ser Ala Ile Ala Ser Gly Val Ala Val Cys Lys Val Leu His Leu

145 150 155 160

Glu Gly Glu Val Asn Lys Ile Lys Ser Ala Leu Leu Ser Thr Asn Lys

165 170 175

Ala Val Val Ser Leu Ser Asn Gly Val Ser Val Leu Thr Phe Lys Val

180 185 190

Leu Asp Leu Lys Asn Tyr Ile Asp Lys Gln Leu Leu Pro Ile Leu Asn

195 200 205

Lys Gln Ser Cys Ser Ile Ser Asn Ile Glu Thr Val Ile Glu Phe Gln

210 215 220

Gln Lys Asn Asn Arg Leu Leu Glu Ile Thr Arg Glu Phe Ser Val Asn

225 230 235 240

Ala Gly Val Thr Thr Pro Val Ser Thr Tyr Met Leu Thr Asn Ser Glu

245 250 255

Leu Leu Ser Leu Ile Asn Asp Met Pro Ile Thr Asn Asp Gln Lys Lys

260 265 270

Leu Met Ser Asn Asn Val Gln Ile Val Arg Gln Gln Ser Tyr Ser Ile

275 280 285

Met Cys Ile Ile Lys Glu Glu Val Leu Ala Tyr Val Val Gln Leu Pro

290 295 300

Leu Tyr Gly Val Ile Asp Thr Pro Cys Trp Lys Leu His Thr Ser Pro

305 310 315 320

Leu Cys Thr Thr Asn Thr Lys Glu Gly Ser Asn Ile Cys Leu Thr Arg

325 330 335

Thr Asp Arg Gly Trp Tyr Cys Asp Asn Ala Gly Ser Val Ser Phe Phe

340 345 350

Pro Gln Ala Glu Thr Cys Lys Val Gln Ser Asn Arg Val Phe Cys Asp

355 360 365

Thr Met Asn Ser Leu Thr Leu Pro Ser Glu Val Asn Leu Cys Asn Val

370 375 380

Asp Ile Phe Asn Pro Lys Tyr Asp Cys Lys Ile Met Thr Ser Lys Thr

385 390 395 400

Asp Val Ser Ser Ser Val Ile Thr Ser Leu Gly Ala Ile Val Ser Cys

405 410 415

Tyr Gly Lys Thr Lys Cys Thr Ala Ser Asn Lys Asn Arg Gly Ile Ile

420 425 430

Lys Thr Phe Ser Asn Gly Cys Asp Tyr Val Ser Asn Lys Gly Val Asp

435 440 445

Thr Val Ser Val Gly Asn Thr Leu Tyr Tyr Val Asn Lys Gln Glu Gly

450 455 460

Lys Ser Leu Tyr Val Lys Gly Glu Pro Ile Ile Asn Phe Tyr Asp Pro

465 470 475 480

Leu Val Phe Pro Ser Asp Glu Phe Asp Ala Ser Ile Ser Gln Val Asn

485 490 495

Glu Lys Ile Asn Gln Ser Leu Ala Phe Ile Arg Lys Ser Asp Glu Leu

500 505 510

Leu Gly Ser Gly Tyr Ile Pro Glu Ala Pro Arg Asp Gly Gln Ala Tyr

515 520 525

Val Arg Lys Asp Gly Glu Trp Val Leu Leu Ser Thr Phe Leu Ile Lys

530 535 540

Leu Glu Leu Glu Lys Ile Lys Leu Glu Leu Glu Lys Ile Lys Leu Glu

545 550 555 560

Leu Glu Lys Asp Tyr Lys Asp Asp Asp Asp Lys

565 570

<210> 46

<211> 571

<212>PRT

<213> Artificial sequence

<220>

<223> F_DS-Cav1-CC07

<400> 46

Met Glu Leu Leu Ile Leu Lys Ala Asn Ala Ile Thr Thr Ile Leu Thr

1 5 10 15

Ala Val Thr Phe Cys Phe Ala Ser Gly Gln Asn Ile Thr Glu Glu Phe

20 25 30

Tyr Gln Ser Thr Cys Ser Ala Val Ser Lys Gly Tyr Leu Ser Ala Leu

35 40 45

Arg Thr Gly Trp Tyr Thr Ser Val Ile Thr Ile Glu Leu Ser Asn Ile

50 55 60

Lys Glu Asn Lys Cys Asn Gly Thr Asp Ala Lys Val Lys Leu Ile Lys

65 70 75 80

Gln Glu Leu Asp Lys Tyr Lys Asn Ala Val Thr Glu Leu Gln Leu Leu

85 90 95

Met Gln Ser Thr Pro Ala Thr Asn Asn Arg Ala Arg Arg Glu Leu Pro

100 105 110

Arg Phe Met Asn Tyr Thr Leu Asn Asn Ala Lys Lys Thr Asn Val Thr

115 120 125

Leu Ser Lys Lys Arg Lys Arg Arg Phe Leu Gly Phe Leu Leu Gly Val

130 135 140

Gly Ser Ala Ile Ala Ser Gly Val Ala Val Cys Lys Val Leu His Leu

145 150 155 160

Glu Gly Glu Val Asn Lys Ile Lys Ser Ala Leu Leu Ser Thr Asn Lys

165 170 175

Ala Val Val Ser Leu Ser Asn Gly Val Ser Val Leu Thr Phe Lys Val

180 185 190

Leu Asp Leu Lys Asn Tyr Ile Asp Lys Gln Leu Leu Pro Ile Leu Asn

195 200 205

Lys Gln Ser Cys Ser Ile Ser Asn Ile Glu Thr Val Ile Glu Phe Gln

210 215 220

Gln Lys Asn Asn Arg Leu Leu Glu Ile Thr Arg Glu Phe Ser Val Asn

225 230 235 240

Ala Gly Val Thr Thr Pro Val Ser Thr Tyr Met Leu Thr Asn Ser Glu

245 250 255

Leu Leu Ser Leu Ile Asn Asp Met Pro Ile Thr Asn Asp Gln Lys Lys

260 265 270

Leu Met Ser Asn Asn Val Gln Ile Val Arg Gln Gln Ser Tyr Ser Ile

275 280 285

Met Cys Ile Ile Lys Glu Glu Val Leu Ala Tyr Val Val Gln Leu Pro

290 295 300

Leu Tyr Gly Val Ile Asp Thr Pro Cys Trp Lys Leu His Thr Ser Pro

305 310 315 320

Leu Cys Thr Thr Asn Thr Lys Glu Gly Ser Asn Ile Cys Leu Thr Arg

325 330 335

Thr Asp Arg Gly Trp Tyr Cys Asp Asn Ala Gly Ser Val Ser Phe Phe

340 345 350

Pro Gln Ala Glu Thr Cys Lys Val Gln Ser Asn Arg Val Phe Cys Asp

355 360 365

Thr Met Asn Ser Leu Thr Leu Pro Ser Glu Val Asn Leu Cys Asn Val

370 375 380

Asp Ile Phe Asn Pro Lys Tyr Asp Cys Lys Ile Met Thr Ser Lys Thr

385 390 395 400

Asp Val Ser Ser Ser Val Ile Thr Ser Leu Gly Ala Ile Val Ser Cys

405 410 415

Tyr Gly Lys Thr Lys Cys Thr Ala Ser Asn Lys Asn Arg Gly Ile Ile

420 425 430

Lys Thr Phe Ser Asn Gly Cys Asp Tyr Val Ser Asn Lys Gly Val Asp

435 440 445

Thr Val Ser Val Gly Asn Thr Leu Tyr Tyr Val Asn Lys Gln Glu Gly

450 455 460

Lys Ser Leu Tyr Val Lys Gly Glu Pro Ile Ile Asn Phe Tyr Asp Pro

465 470 475 480

Leu Val Phe Pro Ser Asp Glu Phe Asp Ala Ser Ile Ser Gln Val Asn

485 490 495

Glu Lys Ile Asn Gln Ser Leu Ala Phe Ile Arg Lys Ser Asp Glu Leu

500 505 510

Leu Gly Ser Gly Tyr Ile Pro Glu Ala Pro Arg Asp Gly Gln Ala Tyr

515 520 525

Val Arg Lys Asp Gly Glu Trp Val Leu Leu Ser Thr Phe Leu Leu His

530 535 540

Leu His Ile Glu Lys Leu His Leu His Ile Glu Lys Leu His Leu His

545 550 555 560

Ile Glu Lys Asp Tyr Lys Asp Asp Asp Asp Lys

565 570

<210> 47

<211> 571

<212>PRT

<213> Artificial sequence

<220>

<223> F_DS-Cav1-CC08

<400> 47

Met Glu Leu Leu Ile Leu Lys Ala Asn Ala Ile Thr Thr Ile Leu Thr

1 5 10 15

Ala Val Thr Phe Cys Phe Ala Ser Gly Gln Asn Ile Thr Glu Glu Phe

20 25 30

Tyr Gln Ser Thr Cys Ser Ala Val Ser Lys Gly Tyr Leu Ser Ala Leu

35 40 45

Arg Thr Gly Trp Tyr Thr Ser Val Ile Thr Ile Glu Leu Ser Asn Ile

50 55 60

Lys Glu Asn Lys Cys Asn Gly Thr Asp Ala Lys Val Lys Leu Ile Lys

65 70 75 80

Gln Glu Leu Asp Lys Tyr Lys Asn Ala Val Thr Glu Leu Gln Leu Leu

85 90 95

Met Gln Ser Thr Pro Ala Thr Asn Asn Arg Ala Arg Arg Glu Leu Pro

100 105 110

Arg Phe Met Asn Tyr Thr Leu Asn Asn Ala Lys Lys Thr Asn Val Thr

115 120 125

Leu Ser Lys Lys Arg Lys Arg Arg Phe Leu Gly Phe Leu Leu Gly Val

130 135 140

Gly Ser Ala Ile Ala Ser Gly Val Ala Val Cys Lys Val Leu His Leu

145 150 155 160

Glu Gly Glu Val Asn Lys Ile Lys Ser Ala Leu Leu Ser Thr Asn Lys

165 170 175

Ala Val Val Ser Leu Ser Asn Gly Val Ser Val Leu Thr Phe Lys Val

180 185 190

Leu Asp Leu Lys Asn Tyr Ile Asp Lys Gln Leu Leu Pro Ile Leu Asn

195 200 205

Lys Gln Ser Cys Ser Ile Ser Asn Ile Glu Thr Val Ile Glu Phe Gln

210 215 220

Gln Lys Asn Asn Arg Leu Leu Glu Ile Thr Arg Glu Phe Ser Val Asn

225 230 235 240

Ala Gly Val Thr Thr Pro Val Ser Thr Tyr Met Leu Thr Asn Ser Glu

245 250 255

Leu Leu Ser Leu Ile Asn Asp Met Pro Ile Thr Asn Asp Gln Lys Lys

260 265 270

Leu Met Ser Asn Asn Val Gln Ile Val Arg Gln Gln Ser Tyr Ser Ile

275 280 285

Met Cys Ile Ile Lys Glu Glu Val Leu Ala Tyr Val Val Gln Leu Pro

290 295 300

Leu Tyr Gly Val Ile Asp Thr Pro Cys Trp Lys Leu His Thr Ser Pro

305 310 315 320

Leu Cys Thr Thr Asn Thr Lys Glu Gly Ser Asn Ile Cys Leu Thr Arg

325 330 335

Thr Asp Arg Gly Trp Tyr Cys Asp Asn Ala Gly Ser Val Ser Phe Phe

340 345 350

Pro Gln Ala Glu Thr Cys Lys Val Gln Ser Asn Arg Val Phe Cys Asp

355 360 365

Thr Met Asn Ser Leu Thr Leu Pro Ser Glu Val Asn Leu Cys Asn Val

370 375 380

Asp Ile Phe Asn Pro Lys Tyr Asp Cys Lys Ile Met Thr Ser Lys Thr

385 390 395 400

Asp Val Ser Ser Ser Val Ile Thr Ser Leu Gly Ala Ile Val Ser Cys

405 410 415

Tyr Gly Lys Thr Lys Cys Thr Ala Ser Asn Lys Asn Arg Gly Ile Ile

420 425 430

Lys Thr Phe Ser Asn Gly Cys Asp Tyr Val Ser Asn Lys Gly Val Asp

435 440 445

Thr Val Ser Val Gly Asn Thr Leu Tyr Tyr Val Asn Lys Gln Glu Gly

450 455 460

Lys Ser Leu Tyr Val Lys Gly Glu Pro Ile Ile Asn Phe Tyr Asp Pro

465 470 475 480

Leu Val Phe Pro Ser Asp Glu Phe Asp Ala Ser Ile Ser Gln Val Asn

485 490 495

Glu Lys Ile Asn Gln Ser Leu Ala Phe Ile Arg Lys Ser Asp Glu Leu

500 505 510

Leu Gly Ser Gly Tyr Ile Pro Glu Ala Pro Arg Asp Gly Gln Ala Tyr

515 520 525

Val Arg Lys Asp Gly Glu Trp Val Leu Leu Ser Thr Phe Leu Leu Lys

530 535 540

Leu Glu Ile His His Leu Lys Leu Glu Ile His His Leu Lys Leu Glu

545 550 555 560

Ile His His Asp Tyr Lys Asp Asp Asp Asp Lys

565 570

<210> 48

<211> 571

<212>PRT

<213> Artificial sequence

<220>

<223> FH_82-CC07

<400> 48

Met Glu Leu Leu Ile Leu Lys Ala Asn Ala Ile Thr Thr Ile Leu Thr

1 5 10 15

Ala Val Thr Phe Cys Phe Ala Ser Gly Gln Asn Ile Thr Glu Glu Phe

20 25 30

Tyr Gln Ser Thr Cys Ser Ala Val Ser Arg Gly Tyr Leu Ser Ala Leu

35 40 45

Arg Thr Gly Trp Tyr Thr Ser Val Ile Thr Ile Glu Leu Ser Asn Ile

50 55 60

Lys Glu Asn Lys Cys Asn Gly Thr Asp Ala Lys Val Lys Leu Ile Lys

65 70 75 80

Gln Glu Leu Asp Lys Tyr Lys Asn Ala Val Thr Glu Leu Gln Leu Leu

85 90 95

Met Gln Ser Thr Pro Ala Thr Asn Asn Arg Ala Arg Arg Glu Leu Pro

100 105 110

Arg Phe Met Asn Tyr Thr Leu Asn Asn Ala Lys Lys Thr Asn Val Thr

115 120 125

Leu Ser Lys Lys Arg Lys Asn Asn Phe Leu Gly Phe Cys Leu Gly Val

130 135 140

Gly Ser Ala Ile Ala Ser Gly Val Ala Val Ser Lys Val Leu His Leu

145 150 155 160

Glu Gly Glu Val Asn Lys Ile Lys Ser Ala Leu Leu Ser Thr Asn Lys

165 170 175

Ala Val Val Ser Leu Ser Asn Gly Val Ser Val Leu Val Val Lys Val

180 185 190

Leu Asp Leu Lys Asn Tyr Ile Asp Lys Gln Leu Leu Pro Ile Val Asn

195 200 205

Lys Gln Ser Cys Ser Ile Ser Asn Ile Glu Thr Val Ile Glu Phe Gln

210 215 220

Gln Lys Asn Asn Arg Leu Leu Glu Ile Thr Arg Glu Phe Ser Val Asn

225 230 235 240

Ala Gly Val Thr Thr Pro Val Ser Thr Tyr Met Leu Thr Asn Ser Glu

245 250 255

Leu Leu Ser Leu Ile Asn Asp Met Pro Ile Thr Asn Asp Gln Lys Lys

260 265 270

Leu Met Ser Asn Asn Val Gln Ile Val Arg Gln Gln Ser Tyr Ser Ile

275 280 285

Met Ser Ile Ile Lys Glu Glu Val Leu Ala Tyr Val Val Gln Leu Pro

290 295 300

Leu Tyr Gly Val Ile Asp Thr Pro Cys Trp Lys Leu His Thr Ser Pro

305 310 315 320

Leu Cys Thr Thr Asn Thr Lys Glu Gly Ser Asn Ile Cys Leu Thr Arg

325 330 335

Thr Asp Arg Gly Trp Tyr Cys Asp Asn Ala Gly Ser Val Ser Phe Phe

340 345 350

Pro Gln Ala Glu Thr Cys Lys Val Gln Ser Asn Arg Val Phe Cys Asp

355 360 365

Thr Met Asn Ser Cys Thr Leu Pro Ser Glu Val Asn Leu Cys Asn Thr

370 375 380

Asp Ile Phe Asn Pro Lys Tyr Asp Cys Lys Ile Met Thr Ser Lys Thr

385 390 395 400

Asp Val Ser Ser Ser Val Ile Thr Ser Leu Gly Ala Ile Val Ser Cys

405 410 415

Tyr Gly Lys Thr Lys Cys Thr Ala Ser Asn Lys Asn Arg Gly Ile Ile

420 425 430

Lys Thr Phe Ser Asn Gly Cys Asp Tyr Val Ser Asn Lys Gly Val Asp

435 440 445

Thr Val Ser Val Gly Asn Thr Leu Tyr Tyr Val Asn Lys Gln Glu Gly

450 455 460

Lys Ser Leu Tyr Val Lys Gly Glu Pro Ile Ile Asn Phe Tyr Asp Pro

465 470 475 480

Leu Val Phe Pro Ser Asp Glu Phe Asp Ala Ser Ile Ser Gln Val Asn

485 490 495

Glu Lys Ile Asn Gln Ser Leu Ala Phe Ile Arg Lys Ser Asp Glu Leu

500 505 510

Leu Gly Ser Gly Tyr Ile Pro Glu Ala Pro Arg Asp Gly Gln Ala Tyr

515 520 525

Val Arg Lys Asp Gly Glu Trp Val Leu Leu Ser Thr Phe Leu Leu His

530 535 540

Leu His Ile Glu Lys Leu His Leu His Ile Glu Lys Leu His Leu His

545 550 555 560

Ile Glu Lys Asp Tyr Lys Asp Asp Asp Asp Lys

565 570

<210> 49

<211> 571

<212>PRT

<213> Artificial sequence

<220>

<223> FH_85-CC07

<400> 49

Met Glu Leu Leu Ile Leu Lys Ala Asn Ala Ile Thr Thr Ile Leu Thr

1 5 10 15

Ala Val Thr Phe Cys Phe Ala Ser Gly Gln Asn Ile Thr Glu Glu Phe

20 25 30

Tyr Gln Ser Thr Cys Ser Ala Val Ser Arg Gly Tyr Leu Ser Ala Leu

35 40 45

Arg Thr Gly Trp Tyr Thr Ser Val Ile Thr Ile Met Leu Ser Asn Ile

50 55 60

Lys Glu Asn Lys Cys Asn Gly Thr Asp Ala Lys Val Lys Leu Ile Lys

65 70 75 80

Gln Glu Leu Asp Lys Tyr Lys Asn Ala Val Thr Glu Leu Gln Leu Leu

85 90 95

Met Gln Ser Thr Pro Ala Thr Asn Asn Arg Ala Arg Arg Glu Leu Pro

100 105 110

Arg Phe Met Asn Tyr Thr Leu Asn Asn Ala Lys Lys Thr Asn Val Thr

115 120 125

Leu Ser Lys Lys Arg Lys Asn Asn Phe Leu Gly Phe Cys Leu Gly Val

130 135 140

Gly Ser Ala Ile Ala Ser Gly Val Ala Val Ser Lys Val Leu His Leu

145 150 155 160

Glu Gly Glu Val Asn Lys Ile Lys Ser Ala Leu Leu Ser Thr Asn Lys

165 170 175

Ala Val Val Ser Leu Ser Asn Gly Val Ser Val Leu Val Val Lys Val

180 185 190

Leu Asp Leu Lys Asn Tyr Ile Asp Lys Gln Leu Leu Pro Ile Val Asn

195 200 205

Lys Gln Ser Cys Ser Ile Ser Asn Ile Glu Thr Val Ile Glu Phe Gln

210 215 220

Gln Lys Asn Asn Arg Leu Leu Glu Ile Thr Arg Glu Phe Ser Val Asn

225 230 235 240

Ala Gly Val Thr Thr Pro Val Ser Thr Tyr Met Leu Thr Asn Ser Glu

245 250 255

Leu Leu Ser Leu Ile Asn Asp Met Pro Ile Thr Asn Asp Gln Lys Lys

260 265 270

Leu Met Ser Asn Asn Val Gln Ile Val Arg Gln Gln Ser Tyr Ser Ile

275 280 285

Met Ser Ile Ile Lys Glu Glu Val Leu Ala Tyr Val Val Gln Leu Pro

290 295 300

Leu Tyr Gly Val Ile Asp Thr Pro Cys Trp Lys Leu His Thr Ser Pro

305 310 315 320

Leu Cys Thr Thr Asn Thr Lys Glu Gly Ser Asn Ile Cys Leu Thr Arg

325 330 335

Thr Asp Arg Gly Trp Tyr Cys Asp Asn Ala Gly Ser Val Ser Phe Phe

340 345 350

Pro Gln Ala Glu Thr Cys Lys Val Gln Ser Asn Arg Val Phe Cys Asp

355 360 365

Thr Met Asn Ser Cys Thr Leu Pro Ser Glu Val Asn Leu Cys Asn Thr

370 375 380

Asp Ile Phe Asn Pro Lys Tyr Asp Cys Lys Ile Met Thr Ser Lys Thr

385 390 395 400

Asp Val Ser Ser Ser Val Ile Thr Ser Leu Gly Ala Ile Val Ser Cys

405 410 415

Tyr Gly Lys Thr Lys Cys Thr Ala Ser Asn Lys Asn Arg Gly Ile Ile

420 425 430

Lys Thr Phe Ser Asn Gly Cys Asp Tyr Val Ser Asn Lys Gly Val Asp

435 440 445

Thr Val Ser Val Gly Asn Thr Leu Tyr Tyr Val Asn Lys Gln Glu Gly

450 455 460

Lys Ser Leu Tyr Val Lys Gly Glu Pro Ile Ile Asn Phe Tyr Asp Pro

465 470 475 480

Leu Val Phe Pro Ser Asp Glu Phe Asp Ala Ser Ile Ser Gln Val Asn

485 490 495

Glu Lys Ile Asn Gln Ser Leu Ala Phe Ile Arg Lys Ser Asp Glu Leu

500 505 510

Leu Gly Ser Gly Tyr Ile Pro Glu Ala Pro Arg Asp Gly Gln Ala Tyr

515 520 525

Val Arg Lys Asp Gly Glu Trp Val Leu Leu Ser Thr Phe Leu Leu His

530 535 540

Leu His Ile Glu Lys Leu His Leu His Ile Glu Lys Leu His Leu His

545 550 555 560

Ile Glu Lys Asp Tyr Lys Asp Asp Asp Asp Lys

565 570

<210> 50

<211> 149

<212>PRT

<213> Artificial sequence

<220>

<223> Self-associating domain of HBcAg

<400> 50

Met Asp Ile Asp Pro Tyr Lys Glu Phe Gly Ala Ser Val Glu Leu Leu

1 5 10 15

Ser Phe Leu Pro Ser Asp Phe Phe Pro Ser Ile Arg Asp Leu Leu Asp

20 25 30

Thr Ala Ser Ala Leu Tyr Arg Glu Ala Leu Glu Ser Pro Glu His Cys

35 40 45

Ser Pro His His Thr Ala Leu Arg Gln Ala Ile Leu Cys Trp Gly Glu

50 55 60

Leu Met Asn Leu Ala Thr Trp Val Gly Ser Asn Leu Glu Asp Pro Ala

65 70 75 80

Ser Arg Glu Leu Val Val Ser Tyr Val Asn Val Asn Met Gly Leu Lys

85 90 95

Ile Arg Gln Leu Leu Trp Phe His Ile Ser Cys Leu Thr Phe Gly Arg

100 105 110

Glu Thr Val Leu Glu Tyr Leu Val Ser Phe Gly Val Trp Ile Arg Thr

115 120 125

Pro Pro Ala Ala Arg Pro Pro Asn Ala Pro Ile Leu Ser Thr Leu Pro

130 135 140

Glu Thr Thr Val Val

145

<210> 51

<211> 175

<212>PRT

<213> Artificial sequence

<220>

<223> GS+self-associating domain HBcAg+His

<400> 51

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

1 5 10 15

Gly Gly Gly Ser Met Asp Ile Asp Pro Tyr Lys Glu Phe Gly Ala Ser

20 25 30

Val Glu Leu Leu Ser Phe Leu Pro Ser Asp Phe Phe Pro Ser Ile Arg

35 40 45

Asp Leu Leu Asp Thr Ala Ser Ala Leu Tyr Arg Glu Ala Leu Glu Ser

50 55 60

Pro Glu His Cys Ser Pro His His Thr Ala Leu Arg Gln Ala Ile Leu

65 70 75 80

Cys Trp Gly Glu Leu Met Asn Leu Ala Thr Trp Val Gly Ser Asn Leu

85 90 95

Glu Asp Pro Ala Ser Arg Glu Leu Val Val Ser Tyr Val Asn Val Asn

100 105 110

Met Gly Leu Lys Ile Arg Gln Leu Leu Trp Phe His Ile Ser Cys Leu

115 120 125

Thr Phe Gly Arg Glu Thr Val Leu Glu Tyr Leu Val Ser Phe Gly Val

130 135 140

Trp Ile Arg Thr Pro Pro Ala Ala Arg Pro Pro Asn Ala Pro Ile Leu

145 150 155 160

Ser Thr Leu Pro Glu Thr Thr Val Val His His His His His

165 170 175

<210> 52

<211> 70

<212>PRT

<213> Artificial sequence

<220>

<223> C-terminal side of the HBcAg self-associating domain

<400> 52

Ala Ser Arg Glu Leu Val Val Ser Tyr Val Asn Val Asn Met Gly Leu

1 5 10 15

Lys Ile Arg Gln Leu Leu Trp Phe His Ile Ser Ala Leu Thr Phe Gly

20 25 30

Arg Glu Thr Val Leu Glu Tyr Leu Val Ser Phe Gly Val Trp Ile Arg

35 40 45

Thr Pro Pro Ala Ala Arg Pro Pro Asn Ala Pro Ile Leu Ser Thr Leu

50 55 60

Pro Glu Thr Thr Val Val

65 70

<210> 53

<211> 65

<212>PRT

<213> Artificial sequence

<220>

<223> N-terminal side of the HBcAg self-associating domain

<400> 53

Gly Ala Ser Val Glu Leu Leu Ser Phe Leu Pro Ser Asp Phe Phe Pro

1 5 10 15

Ser Ile Arg Asp Leu Leu Asp Thr Ala Ser Ala Leu Tyr Arg Glu Ala

20 25 30

Leu Glu Ser Pro Glu His Ser Ser Pro His His Thr Ala Leu Arg Gln

35 40 45

Ala Ile Leu Cys Trp Gly Glu Leu Met Asn Leu Ala Thr Trp Val Gly

50 55 60

Ser

65

<210> 54

<211> 186

<212>PRT

<213> Artificial sequence

<220>

<223> HBcCN

<400> 54

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

1 5 10 15

Gly Gly Gly Ser Ala Ser Arg Glu Leu Val Val Ser Tyr Val Asn Val

20 25 30

Asn Met Gly Leu Lys Ile Arg Gln Leu Leu Trp Phe His Ile Ser Ala

35 40 45

Leu Thr Phe Gly Arg Glu Thr Val Leu Glu Tyr Leu Val Ser Phe Gly

50 55 60

Val Trp Ile Arg Thr Pro Pro Ala Ala Arg Pro Pro Asn Ala Pro Ile

65 70 75 80

Leu Ser Thr Leu Pro Glu Thr Thr Val Val Gly Gly Gly Gly Ser Gly

85 90 95

Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Ala

100 105 110

Ser Val Glu Leu Leu Ser Phe Leu Pro Ser Asp Phe Phe Pro Ser Ile

115 120 125

Arg Asp Leu Leu Asp Thr Ala Ser Ala Leu Tyr Arg Glu Ala Leu Glu

130 135 140

Ser Pro Glu His Ser Ser Pro His His Thr Ala Leu Arg Gln Ala Ile

145 150 155 160

Leu Cys Trp Gly Glu Leu Met Asn Leu Ala Thr Trp Val Gly Ser Gly

165 170 175

Gly Ser Asp Tyr Lys Asp Asp Asp Asp Lys

180 185

<210> 55

<211> 728

<212>PRT

<213> Artificial sequence

<220>

<223> F_DC-Cav1-HBcCN

<400> 55

Met Glu Leu Leu Ile Leu Lys Ala Asn Ala Ile Thr Thr Ile Leu Thr

1 5 10 15

Ala Val Thr Phe Cys Phe Ala Ser Gly Gln Asn Ile Thr Glu Glu Phe

20 25 30

Tyr Gln Ser Thr Cys Ser Ala Val Ser Lys Gly Tyr Leu Ser Ala Leu

35 40 45

Arg Thr Gly Trp Tyr Thr Ser Val Ile Thr Ile Glu Leu Ser Asn Ile

50 55 60

Lys Glu Asn Lys Cys Asn Gly Thr Asp Ala Lys Val Lys Leu Ile Lys

65 70 75 80

Gln Glu Leu Asp Lys Tyr Lys Asn Ala Val Thr Glu Leu Gln Leu Leu

85 90 95

Met Gln Ser Thr Pro Ala Thr Asn Asn Arg Ala Arg Arg Glu Leu Pro

100 105 110

Arg Phe Met Asn Tyr Thr Leu Asn Asn Ala Lys Lys Thr Asn Val Thr

115 120 125

Leu Ser Lys Lys Arg Lys Arg Arg Phe Leu Gly Phe Leu Leu Gly Val

130 135 140

Gly Ser Ala Ile Ala Ser Gly Val Ala Val Cys Lys Val Leu His Leu

145 150 155 160

Glu Gly Glu Val Asn Lys Ile Lys Ser Ala Leu Leu Ser Thr Asn Lys

165 170 175

Ala Val Val Ser Leu Ser Asn Gly Val Ser Val Leu Thr Phe Lys Val

180 185 190

Leu Asp Leu Lys Asn Tyr Ile Asp Lys Gln Leu Leu Pro Ile Leu Asn

195 200 205

Lys Gln Ser Cys Ser Ile Ser Asn Ile Glu Thr Val Ile Glu Phe Gln

210 215 220

Gln Lys Asn Asn Arg Leu Leu Glu Ile Thr Arg Glu Phe Ser Val Asn

225 230 235 240

Ala Gly Val Thr Thr Pro Val Ser Thr Tyr Met Leu Thr Asn Ser Glu

245 250 255

Leu Leu Ser Leu Ile Asn Asp Met Pro Ile Thr Asn Asp Gln Lys Lys

260 265 270

Leu Met Ser Asn Asn Val Gln Ile Val Arg Gln Gln Ser Tyr Ser Ile

275 280 285

Met Cys Ile Ile Lys Glu Glu Val Leu Ala Tyr Val Val Gln Leu Pro

290 295 300

Leu Tyr Gly Val Ile Asp Thr Pro Cys Trp Lys Leu His Thr Ser Pro

305 310 315 320

Leu Cys Thr Thr Asn Thr Lys Glu Gly Ser Asn Ile Cys Leu Thr Arg

325 330 335

Thr Asp Arg Gly Trp Tyr Cys Asp Asn Ala Gly Ser Val Ser Phe Phe

340 345 350

Pro Gln Ala Glu Thr Cys Lys Val Gln Ser Asn Arg Val Phe Cys Asp

355 360 365

Thr Met Asn Ser Leu Thr Leu Pro Ser Glu Val Asn Leu Cys Asn Val

370 375 380

Asp Ile Phe Asn Pro Lys Tyr Asp Cys Lys Ile Met Thr Ser Lys Thr

385 390 395 400

Asp Val Ser Ser Ser Val Ile Thr Ser Leu Gly Ala Ile Val Ser Cys

405 410 415

Tyr Gly Lys Thr Lys Cys Thr Ala Ser Asn Lys Asn Arg Gly Ile Ile

420 425 430

Lys Thr Phe Ser Asn Gly Cys Asp Tyr Val Ser Asn Lys Gly Val Asp

435 440 445

Thr Val Ser Val Gly Asn Thr Leu Tyr Tyr Val Asn Lys Gln Glu Gly

450 455 460

Lys Ser Leu Tyr Val Lys Gly Glu Pro Ile Ile Asn Phe Tyr Asp Pro

465 470 475 480

Leu Val Phe Pro Ser Asp Glu Phe Asp Ala Ser Ile Ser Gln Val Asn

485 490 495

Glu Lys Ile Asn Gln Ser Leu Ala Phe Ile Arg Lys Ser Asp Glu Leu

500 505 510

Leu Gly Ser Gly Tyr Ile Pro Glu Ala Pro Arg Asp Gly Gln Ala Tyr

515 520 525

Val Arg Lys Asp Gly Glu Trp Val Leu Leu Ser Thr Phe Leu Gly Gly

530 535 540

Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly

545 550 555 560

Gly Ser Ala Ser Arg Glu Leu Val Val Ser Tyr Val Asn Val Asn Met

565 570 575

Gly Leu Lys Ile Arg Gln Leu Leu Trp Phe His Ile Ser Ala Leu Thr

580 585 590

Phe Gly Arg Glu Thr Val Leu Glu Tyr Leu Val Ser Phe Gly Val Trp

595 600 605

Ile Arg Thr Pro Pro Ala Ala Arg Pro Pro Asn Ala Pro Ile Leu Ser

610 615 620

Thr Leu Pro Glu Thr Thr Val Val Gly Gly Gly Gly Ser Gly Gly Gly

625 630 635 640

Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Ala Ser Val

645 650 655

Glu Leu Leu Ser Phe Leu Pro Ser Asp Phe Phe Pro Ser Ile Arg Asp

660 665 670

Leu Leu Asp Thr Ala Ser Ala Leu Tyr Arg Glu Ala Leu Glu Ser Pro

675 680 685

Glu His Ser Ser Pro His His Thr Ala Leu Arg Gln Ala Ile Leu Cys

690 695 700

Trp Gly Glu Leu Met Asn Leu Ala Thr Trp Val Gly Ser Gly Gly Ser

705 710 715 720

Asp Tyr Lys Asp Asp Asp Asp Lys

725

<210> 56

<211> 728

<212>PRT

<213> Artificial sequence

<220>

<223> FH_85-HBcCN

<400> 56

Met Glu Leu Leu Ile Leu Lys Ala Asn Ala Ile Thr Thr Ile Leu Thr

1 5 10 15

Ala Val Thr Phe Cys Phe Ala Ser Gly Gln Asn Ile Thr Glu Glu Phe

20 25 30

Tyr Gln Ser Thr Cys Ser Ala Val Ser Arg Gly Tyr Leu Ser Ala Leu

35 40 45

Arg Thr Gly Trp Tyr Thr Ser Val Ile Thr Ile Met Leu Ser Asn Ile

50 55 60

Lys Glu Asn Lys Cys Asn Gly Thr Asp Ala Lys Val Lys Leu Ile Lys

65 70 75 80

Gln Glu Leu Asp Lys Tyr Lys Asn Ala Val Thr Glu Leu Gln Leu Leu

85 90 95

Met Gln Ser Thr Pro Ala Thr Asn Asn Arg Ala Arg Arg Glu Leu Pro

100 105 110

Arg Phe Met Asn Tyr Thr Leu Asn Asn Ala Lys Lys Thr Asn Val Thr

115 120 125

Leu Ser Lys Lys Arg Lys Asn Asn Phe Leu Gly Phe Cys Leu Gly Val

130 135 140

Gly Ser Ala Ile Ala Ser Gly Val Ala Val Ser Lys Val Leu His Leu

145 150 155 160

Glu Gly Glu Val Asn Lys Ile Lys Ser Ala Leu Leu Ser Thr Asn Lys

165 170 175

Ala Val Val Ser Leu Ser Asn Gly Val Ser Val Leu Val Val Lys Val

180 185 190

Leu Asp Leu Lys Asn Tyr Ile Asp Lys Gln Leu Leu Pro Ile Val Asn

195 200 205

Lys Gln Ser Cys Ser Ile Ser Asn Ile Glu Thr Val Ile Glu Phe Gln

210 215 220

Gln Lys Asn Asn Arg Leu Leu Glu Ile Thr Arg Glu Phe Ser Val Asn

225 230 235 240

Ala Gly Val Thr Thr Pro Val Ser Thr Tyr Met Leu Thr Asn Ser Glu

245 250 255

Leu Leu Ser Leu Ile Asn Asp Met Pro Ile Thr Asn Asp Gln Lys Lys

260 265 270

Leu Met Ser Asn Asn Val Gln Ile Val Arg Gln Gln Ser Tyr Ser Ile

275 280 285

Met Ser Ile Ile Lys Glu Glu Val Leu Ala Tyr Val Val Gln Leu Pro

290 295 300

Leu Tyr Gly Val Ile Asp Thr Pro Cys Trp Lys Leu His Thr Ser Pro

305 310 315 320

Leu Cys Thr Thr Asn Thr Lys Glu Gly Ser Asn Ile Cys Leu Thr Arg

325 330 335

Thr Asp Arg Gly Trp Tyr Cys Asp Asn Ala Gly Ser Val Ser Phe Phe

340 345 350

Pro Gln Ala Glu Thr Cys Lys Val Gln Ser Asn Arg Val Phe Cys Asp

355 360 365

Thr Met Asn Ser Cys Thr Leu Pro Ser Glu Val Asn Leu Cys Asn Thr

370 375 380

Asp Ile Phe Asn Pro Lys Tyr Asp Cys Lys Ile Met Thr Ser Lys Thr

385 390 395 400

Asp Val Ser Ser Ser Val Ile Thr Ser Leu Gly Ala Ile Val Ser Cys

405 410 415

Tyr Gly Lys Thr Lys Cys Thr Ala Ser Asn Lys Asn Arg Gly Ile Ile

420 425 430

Lys Thr Phe Ser Asn Gly Cys Asp Tyr Val Ser Asn Lys Gly Val Asp

435 440 445

Thr Val Ser Val Gly Asn Thr Leu Tyr Tyr Val Asn Lys Gln Glu Gly

450 455 460

Lys Ser Leu Tyr Val Lys Gly Glu Pro Ile Ile Asn Phe Tyr Asp Pro

465 470 475 480

Leu Val Phe Pro Ser Asp Glu Phe Asp Ala Ser Ile Ser Gln Val Asn

485 490 495

Glu Lys Ile Asn Gln Ser Leu Ala Phe Ile Arg Lys Ser Asp Glu Leu

500 505 510

Leu Gly Ser Gly Tyr Ile Pro Glu Ala Pro Arg Asp Gly Gln Ala Tyr

515 520 525

Val Arg Lys Asp Gly Glu Trp Val Leu Leu Ser Thr Phe Leu Gly Gly

530 535 540

Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly

545 550 555 560

Gly Ser Ala Ser Arg Glu Leu Val Val Ser Tyr Val Asn Val Asn Met

565 570 575

Gly Leu Lys Ile Arg Gln Leu Leu Trp Phe His Ile Ser Ala Leu Thr

580 585 590

Phe Gly Arg Glu Thr Val Leu Glu Tyr Leu Val Ser Phe Gly Val Trp

595 600 605

Ile Arg Thr Pro Pro Ala Ala Arg Pro Pro Asn Ala Pro Ile Leu Ser

610 615 620

Thr Leu Pro Glu Thr Thr Val Val Gly Gly Gly Gly Ser Gly Gly Gly

625 630 635 640

Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Ala Ser Val

645 650 655

Glu Leu Leu Ser Phe Leu Pro Ser Asp Phe Phe Pro Ser Ile Arg Asp

660 665 670

Leu Leu Asp Thr Ala Ser Ala Leu Tyr Arg Glu Ala Leu Glu Ser Pro

675 680 685

Glu His Ser Ser Pro His His Thr Ala Leu Arg Gln Ala Ile Leu Cys

690 695 700

Trp Gly Glu Leu Met Asn Leu Ala Thr Trp Val Gly Ser Gly Gly Ser

705 710 715 720

Asp Tyr Lys Asp Asp Asp Asp Lys

725

<210> 57

<211> 2307

<212> DNA

<213> Artificial sequence

<220>

<223> F_DS-Cav1-Fc

<400> 57

atggaacttc tcatcctgaa agccaatgcc attacgacca ttctgacagc cgtaaccttt 60

tgctttgcaa gtggtcagaa tatcactgag gaattctacc agagtacctg tagcgcagtt 120

tctaaggggt acttgtctgc gcttcgcact ggctggtaca cctcggttat taccattgag 180

ctgtccaaca tcaaagagaa caagtgcaac ggcaccgacg ccaaagtgaa actgattaaa 240

caggagctcg acaaatacaa aaatgctgtg acagagttgc agttgctcat gcagtctacc 300

cctgctacga acaaccgagc aaggcgagaa ctaccaagat tcatgaacta caccctgaat 360

aatgcgaaaa agactaacgt gaccctcagc aagaagagaa agaggcgttt tcttgggttt 420

ttgctgggag ttgggagcgc tatagccagc ggagttgctg tgtgtaaggt attgcaccta 480

gaaggggagg tgaataagat caagagtgcc ctgctgtcca caaacaaagc agtagtgtcg 540

cttagcaatg gggtgtccgt tctgaccttc aaggttctag atctcaaaaa ctacatcgat 600

aagcagcttc tgccaatcct caacaagcag tcttgcagta ttagcaatat cgagacggtg 660

atagagtttc aacagaagaa caatcgtctg ctcgaaatta cacgggagtt tagcgtcaac 720

gcaggagtga ctactcctgt aagcacctac atgttaacaa actccgagct gctatctctg 780

atcaatgaca tgccaattac aaacgaccag aaaaagttaa tgtcaaacaa tgtgcagata 840

gtcaggcaac agtcctattc cattatgtgc atcatcaagg aagaagttct ggcctatgtc 900

gtccaacttc ccttatatgg cgtcatagac acgccctgtt ggaaactgca caccagtcct 960

ttgtgcacta caaacactaa ggaggggtct aacatctgtc tgactaggac agatcgcggg 1020

tggtattgcg acaatgctgg ctcagtgagc tttttcccac aagccgaaac atgcaaagtc 1080

cagtcgaatc gggtgttttg tgacacaatg aactcactga ctcttccttc cgaagtgaac 1140

ttgtgcaatg tcgatatctt caatcccaaa tatgactgca agatcatgac aagtaagacc 1200

gatgtcagca gtagcgtcat tacctccctc ggtgctattg tgtcctgtta cggcaagacc 1260

aaatgtactg cttctaacaa aaatcgcggc attattaaga cattcagcaa cggatgtgac 1320

tatgtctcca ataaaggtgt agacacggtg tctgtgggta ataccctcta ctatgtgaat 1380

aagcaggaag gaaagtcact ctatgtgaaa ggagagccga tcatcaactt ctacgatccc 1440

ctggtgtttc ccagtgatga gttcgacgcc tctatcagcc aggtgaatga aaagatcaac 1500

caatccctgg ccttcatacg gaaatcagat gagctgttag gctcaggcta catacccgaa 1560

gcaccgagag atggtcaagc gtatgtgcgg aaggatggag agtgggtcct tctgtcaact 1620

ttcctggaca aaactcacac ttgtccgcct tgtcccgctc cagagctcct tggcggaccc 1680

tccgttttcc tgtttcctcc gaaacccaag gatacgctga tgatttcacg cacaccagaa 1740

gtgacatgtg tggtggtaga tgtgtcccat gaagatccgg aggtgaagtt taactggtat 1800

gtcgatggtg tggaggttca taacgctaag acgaaaccac gggaggagca gtacaattcc 1860

acctaccgtg tagtctctgt gctgaccgtt ttgcatcagg attggctgaa tggtaaagag 1920

tataagtgca aagtgtccaa caaggctctg ccagccccta tcgaaaagac catcagtaaa 1980

gccaaaggac agcctagaga accccaggtc tatacgctgc caccctctcg ggacgagctg 2040

accaagaatc aggtgtcact gacttgtctg gtgaagggct tctaccctag cgacattgcc 2100

gtcgaatggg agtctaatgg gcaacccgag aataactaca aaaccacacc gcccgtcttg 2160

gacagcgatg gcagtttctt cctgtactcc aaactgactg tggacaaaag ccggtggcag 2220

caaggcaatg tgttctcatg ctctgtcatg cacgaagccc tgcacaacca ttacacccaa 2280

aagtcactga gcctgagccc tggaaag 2307

<210> 58

<211> 2307

<212> DNA

<213> Artificial sequence

<220>

<223> FH_82-Fc

<400> 58

atggaactgc tgatacttaa ggctaacgcg ataactacga tcctcaccgc cgtgaccttt 60

tgctttgcct ctggccaaaa cattactgag gaattctacc agtcaacgtg cagtgcagtg 120

agccgaggt atctgtccgc cctgagaacc gggtggtata cttccgtcat taccatcgag 180

ctgtccaaca taaaagagaa taagtgcaac ggcaccgatg ctaaagtgaa actgatcaaa 240

caggaactcg ataaatacaa gaatgcagtt acagagcttc agctcctgat gcagagcact 300

ccggccacca ataatagggc aaggagagaa ttgccacgat ttatgaatta cacactcaac 360

aacgcgaaga aaactaacgt gactctgtcc aagaaacgta agaataactt cttggggttc 420

tgtctgggtg taggtagcgc cattgcttct ggggtggccg tcagcaaagt gcttcacctg 480

gaaggagagg tgaacaagat caagtctgca ctgctgtcta caaacaaagc agtggtgagc 540

ctgtccaacg gagtatccgt tctggtggtc aaagtcctgg atctgaagaa ttatatcgac 600

aaacaactgc tccccattgt gaacaagcag agttgttcaa tcagcaacat agaaactgtg 660

attgagttcc aacagaagaa caataggctg ctcgaaatta ccagagagtt tagcgtcaat 720

gctggtgtca caaccccagt cagcacttac atgctgacta attccgagtt gcttagcctt 780

attaacgaca tgcctatcac caatgaccag aagaagctga tgagtaataa tgtgcagatt 840

gtgcgccagc agagttacag cattatgagt attatcaaag aggaggtatt ggcttatgtg 900

gttcagcttc cgctgtatgg ggtcatcgac acaccttgtt ggaagttgca taccagtccc 960

ctgtgtacga caaacaccaa ggaaggtagt aacatctgct tgacacgtac cgatcggggt 1020

tggtattgcg ataacgccgg gtctgttagt ttctttcctc aagccgagac atgcaaagtc 1080

cagagcaatc gcgtgttctg tgacacgatg aacagctgta ctttgccatc agaggttaat 1140

ctgtgcaata ccgacatctt caaccccaaa tacgactgta agatcatgac cagcaagact 1200

gatgtcagct cctccgttat aacatcactc ggcgctatcg tgtcttgcta tggcaagacc 1260

aagtgtacag cgtccaataa gaatcggggc attatcaaga cattctccaa cggatgtgac 1320

tacgtgagca acaaaggagt ggacaccgtg tcagtcggaa atacactgta ttacgtgaat 1380

aagcaggagg gcaaatctct ttacgtgaag ggcgaaccaa tcatcaactt ctatgatccc 1440

ctcgtctttc cttctgatga gtttgacgcc tctatttctc aggttaacga gaagatcaat 1500

cagtctctgg cctttatacg caaaagcgat gaactcctgg gctcaggcta catacccgaa 1560

gcaccgagag atggtcaagc gtatgtgcgg aaggatggag agtgggtcct tctgtcaact 1620

ttcctggaca aaactcacac ttgtccgcct tgtcccgctc cagagctcct tggcggaccc 1680

tccgttttcc tgtttcctcc gaaacccaag gatacgctga tgatttcacg cacaccagaa 1740

gtgacatgtg tggtggtaga tgtgtcccat gaagatccgg aggtgaagtt taactggtat 1800

gtcgatggtg tggaggttca taacgctaag acgaaaccac gggaggagca gtacaattcc 1860

acctaccgtg tagtctctgt gctgaccgtt ttgcatcagg attggctgaa tggtaaagag 1920

tataagtgca aagtgtccaa caaggctctg ccagccccta tcgaaaagac catcagtaaa 1980

gccaaaggac agcctagaga accccaggtc tatacgctgc caccctctcg ggacgagctg 2040

accaagaatc aggtgtcact gacttgtctg gtgaagggct tctaccctag cgacattgcc 2100

gtcgaatggg agtctaatgg gcaacccgag aataactaca aaaccacacc gcccgtcttg 2160

gacagcgatg gcagtttctt cctgtactcc aaactgactg tggacaaaag ccggtggcag 2220

caaggcaatg tgttctcatg ctctgtcatg cacgaagccc tgcacaacca ttacacccaa 2280

aagtcactga gcctgagccc tggaaag 2307

<210> 59

<211> 2307

<212> DNA

<213> Artificial sequence

<220>

<223> FH_85-Fc

<400> 59

atggaactgc tgatacttaa ggctaacgcg ataactacga tcctcaccgc cgtgaccttt 60

tgctttgcct ctggccaaaa cattactgag gaattctacc agtcaacgtg cagtgcagtg 120

agccgaggt atctgtccgc cctgagaacc gggtggtata cttccgtcat taccatcatg 180

ctgtccaaca taaaagagaa taagtgcaac ggcaccgatg ctaaagtgaa actgatcaaa 240

caggaactcg ataaatacaa gaatgcagtt acagagcttc agctcctgat gcagagcact 300

ccggccacca ataatagggc aaggagagaa ttgccacgat ttatgaatta cacactcaac 360

aacgcgaaga aaactaacgt gactctgtcc aagaaacgta agaataactt cttggggttc 420

tgtctgggtg taggtagcgc cattgcttct ggggtggccg tcagcaaagt gcttcacctg 480

gaaggagagg tgaacaagat caagtctgca ctgctgtcta caaacaaagc agtggtgagc 540

ctgtccaacg gagtatccgt tctggtggtc aaagtcctgg atctgaagaa ttatatcgac 600

aaacaactgc tccccattgt gaacaagcag agttgttcaa tcagcaacat agaaactgtg 660

attgagttcc aacagaagaa caataggctg ctcgaaatta ccagagagtt tagcgtcaat 720

gctggtgtca caaccccagt cagcacttac atgctgacta attccgagtt gcttagcctt 780

attaacgaca tgcctatcac caatgaccag aagaagctga tgagtaataa tgtgcagatt 840

gtgcgccagc agagttacag cattatgagt attatcaaag aggaggtatt ggcttatgtg 900

gttcagcttc cgctgtatgg ggtcatcgac acaccttgtt ggaagttgca taccagtccc 960

ctgtgtacga caaacaccaa ggaaggtagt aacatctgct tgacacgtac cgatcggggt 1020

tggtattgcg ataacgccgg gtctgttagt ttctttcctc aagccgagac atgcaaagtc 1080

cagagcaatc gcgtgttctg tgacacgatg aacagctgta ctttgccatc agaggttaat 1140

ctgtgcaata ccgacatctt caaccccaaa tacgactgta agatcatgac cagcaagact 1200

gatgtcagct cctccgttat aacatcactc ggcgctatcg tgtcttgcta tggcaagacc 1260

aagtgtacag cgtccaataa gaatcggggc attatcaaga cattctccaa cggatgtgac 1320

tacgtgagca acaaaggagt ggacaccgtg tcagtcggaa atacactgta ttacgtgaat 1380

aagcaggagg gcaaatctct ttacgtgaag ggcgaaccaa tcatcaactt ctatgatccc 1440

ctcgtctttc cttctgatga gtttgacgcc tctatttctc aggttaacga gaagatcaat 1500

cagtctctgg cctttatacg caaaagcgat gaactcctgg gctcaggcta catacccgaa 1560

gcaccgagag atggtcaagc gtatgtgcgg aaggatggag agtgggtcct tctgtcaact 1620

ttcctggaca aaactcacac ttgtccgcct tgtcccgctc cagagctcct tggcggaccc 1680

tccgttttcc tgtttcctcc gaaacccaag gatacgctga tgatttcacg cacaccagaa 1740

gtgacatgtg tggtggtaga tgtgtcccat gaagatccgg aggtgaagtt taactggtat 1800

gtcgatggtg tggaggttca taacgctaag acgaaaccac gggaggagca gtacaattcc 1860

acctaccgtg tagtctctgt gctgaccgtt ttgcatcagg attggctgaa tggtaaagag 1920

tataagtgca aagtgtccaa caaggctctg ccagccccta tcgaaaagac catcagtaaa 1980

gccaaaggac agcctagaga accccaggtc tatacgctgc caccctctcg ggacgagctg 2040

accaagaatc aggtgtcact gacttgtctg gtgaagggct tctaccctag cgacattgcc 2100

gtcgaatggg agtctaatgg gcaacccgag aataactaca aaaccacacc gcccgtcttg 2160

gacagcgatg gcagtttctt cctgtactcc aaactgactg tggacaaaag ccggtggcag 2220

caaggcaatg tgttctcatg ctctgtcatg cacgaagccc tgcacaacca ttacacccaa 2280

aagtcactga gcctgagccc tggaaag 2307

<210> 60

<211> 2367

<212> DNA

<213> Artificial sequence

<220>

<223> F_DS-Cav1-4GS-Fc

<400> 60

atggaacttc tcatcctgaa agccaatgcc attacgacca ttctgacagc cgtaaccttt 60

tgctttgcaa gtggtcagaa tatcactgag gaattctacc agagtacctg tagcgcagtt 120

tctaaggggt acttgtctgc gcttcgcact ggctggtaca cctcggttat taccattgag 180

ctgtccaaca tcaaagagaa caagtgcaac ggcaccgacg ccaaagtgaa actgattaaa 240

caggagctcg acaaatacaa aaatgctgtg acagagttgc agttgctcat gcagtctacc 300

cctgctacga acaaccgagc aaggcgagaa ctaccaagat tcatgaacta caccctgaat 360

aatgcgaaaa agactaacgt gaccctcagc aagaagagaa agaggcgttt tcttgggttt 420

ttgctgggag ttgggagcgc tatagccagc ggagttgctg tgtgtaaggt attgcaccta 480

gaaggggagg tgaataagat caagagtgcc ctgctgtcca caaacaaagc agtagtgtcg 540

cttagcaatg gggtgtccgt tctgaccttc aaggttctag atctcaaaaa ctacatcgat 600

aagcagcttc tgccaatcct caacaagcag tcttgcagta ttagcaatat cgagacggtg 660

atagagtttc aacagaagaa caatcgtctg ctcgaaatta cacgggagtt tagcgtcaac 720

gcaggagtga ctactcctgt aagcacctac atgttaacaa actccgagct gctatctctg 780

atcaatgaca tgccaattac aaacgaccag aaaaagttaa tgtcaaacaa tgtgcagata 840

gtcaggcaac agtcctattc cattatgtgc atcatcaagg aagaagttct ggcctatgtc 900

gtccaacttc ccttatatgg cgtcatagac acgccctgtt ggaaactgca caccagtcct 960

ttgtgcacta caaacactaa ggaggggtct aacatctgtc tgactaggac agatcgcggg 1020

tggtattgcg acaatgctgg ctcagtgagc tttttcccac aagccgaaac atgcaaagtc 1080

cagtcgaatc gggtgttttg tgacacaatg aactcactga ctcttccttc cgaagtgaac 1140

ttgtgcaatg tcgatatctt caatcccaaa tatgactgca agatcatgac aagtaagacc 1200

gatgtcagca gtagcgtcat tacctccctc ggtgctattg tgtcctgtta cggcaagacc 1260

aaatgtactg cttctaacaa aaatcgcggc attattaaga cattcagcaa cggatgtgac 1320

tatgtctcca ataaaggtgt agacacggtg tctgtgggta ataccctcta ctatgtgaat 1380

aagcaggaag gaaagtcact ctatgtgaaa ggagagccga tcatcaactt ctacgatccc 1440

ctggtgtttc ccagtgatga gttcgacgcc tctatcagcc aggtgaatga aaagatcaac 1500

caatccctgg ccttcatacg gaaatcagat gagctgttag gctcaggcta catacccgaa 1560

gcaccgagag atggtcaagc gtatgtgcgg aaggatggag agtgggtcct tctgtcaact 1620

ttcctgggcg ggggtgggag cggaggcgga ggttcagggg gtggagggtc aggcggagga 1680

ggcagcgaca aaactcacac ttgtccgcct tgtcccgctc cagagctcct tggcggaccc 1740

tccgttttcc tgtttcctcc gaaacccaag gatacgctga tgatttcacg cacaccagaa 1800

gtgacatgtg tggtggtaga tgtgtcccat gaagatccgg aggtgaagtt taactggtat 1860

gtcgatggtg tggaggttca taacgctaag acgaaaccac gggaggagca gtacaattcc 1920

acctaccgtg tagtctctgt gctgaccgtt ttgcatcagg attggctgaa tggtaaagag 1980

tataagtgca aagtgtccaa caaggctctg ccagccccta tcgaaaagac catcagtaaa 2040

gccaaaggac agcctagaga accccaggtc tatacgctgc caccctctcg ggacgagctg 2100

accaagaatc aggtgtcact gacttgtctg gtgaagggct tctaccctag cgacattgcc 2160

gtcgaatggg agtctaatgg gcaacccgag aataactaca aaaccacacc gcccgtcttg 2220

gacagcgatg gcagtttctt cctgtactcc aaactgactg tggacaaaag ccggtggcag 2280

caaggcaatg tgttctcatg ctctgtcatg cacgaagccc tgcacaacca ttacacccaa 2340

aagtcactga gcctgagccc tggaaag 2367

<210> 61

<211> 2337

<212> DNA

<213> Artificial sequence

<220>

<223> F_DS-Cav1-2GS-Fc

<400> 61

atggaacttc tcatcctgaa agccaatgcc attacgacca ttctgacagc cgtaaccttt 60

tgctttgcaa gtggtcagaa tatcactgag gaattctacc agagtacctg tagcgcagtt 120

tctaaggggt acttgtctgc gcttcgcact ggctggtaca cctcggttat taccattgag 180

ctgtccaaca tcaaagagaa caagtgcaac ggcaccgacg ccaaagtgaa actgattaaa 240

caggagctcg acaaatacaa aaatgctgtg acagagttgc agttgctcat gcagtctacc 300

cctgctacga acaaccgagc aaggcgagaa ctaccaagat tcatgaacta caccctgaat 360

aatgcgaaaa agactaacgt gaccctcagc aagaagagaa agaggcgttt tcttgggttt 420

ttgctgggag ttgggagcgc tatagccagc ggagttgctg tgtgtaaggt attgcaccta 480

gaaggggagg tgaataagat caagagtgcc ctgctgtcca caaacaaagc agtagtgtcg 540

cttagcaatg gggtgtccgt tctgaccttc aaggttctag atctcaaaaa ctacatcgat 600

aagcagcttc tgccaatcct caacaagcag tcttgcagta ttagcaatat cgagacggtg 660

atagagtttc aacagaagaa caatcgtctg ctcgaaatta cacgggagtt tagcgtcaac 720

gcaggagtga ctactcctgt aagcacctac atgttaacaa actccgagct gctatctctg 780

atcaatgaca tgccaattac aaacgaccag aaaaagttaa tgtcaaacaa tgtgcagata 840

gtcaggcaac agtcctattc cattatgtgc atcatcaagg aagaagttct ggcctatgtc 900

gtccaacttc ccttatatgg cgtcatagac acgccctgtt ggaaactgca caccagtcct 960

ttgtgcacta caaacactaa ggaggggtct aacatctgtc tgactaggac agatcgcggg 1020

tggtattgcg acaatgctgg ctcagtgagc tttttcccac aagccgaaac atgcaaagtc 1080

cagtcgaatc gggtgttttg tgacacaatg aactcactga ctcttccttc cgaagtgaac 1140

ttgtgcaatg tcgatatctt caatcccaaa tatgactgca agatcatgac aagtaagacc 1200

gatgtcagca gtagcgtcat tacctccctc ggtgctattg tgtcctgtta cggcaagacc 1260

aaatgtactg cttctaacaa aaatcgcggc attattaaga cattcagcaa cggatgtgac 1320

tatgtctcca ataaaggtgt agacacggtg tctgtgggta ataccctcta ctatgtgaat 1380

aagcaggaag gaaagtcact ctatgtgaaa ggagagccga tcatcaactt ctacgatccc 1440

ctggtgtttc ccagtgatga gttcgacgcc tctatcagcc aggtgaatga aaagatcaac 1500

caatccctgg ccttcatacg gaaatcagat gagctgttag gctcaggcta catacccgaa 1560

gcaccgagag atggtcaagc gtatgtgcgg aaggatggag agtgggtcct tctgtcaact 1620

ttcctggggg gtggagggtc aggcggagga ggcagcgaca aaactcacac ttgtccgcct 1680

tgtcccgctc cagagctcct tggcggaccc tccgttttcc tgtttcctcc gaaacccaag 1740

gatacgctga tgatttcacg cacaccagaa gtgacatgtg tggtggtaga tgtgtcccat 1800

gaagatccgg aggtgaagtt taactggtat gtcgatggtg tggaggttca taacgctaag 1860

acgaaaccac gggaggagca gtacaattcc acctaccgtg tagtctctgt gctgaccgtt 1920

ttgcatcagg attggctgaa tggtaaagag tataagtgca aagtgtccaa caaggctctg 1980

ccagccccta tcgaaaagac catcagtaaa gccaaaggac agcctagaga accccaggtc 2040

tatacgctgc caccctctcg ggacgagctg accaagaatc aggtgtcact gacttgtctg 2100

gtgaagggct tctaccctag cgacattgcc gtcgaatggg agtctaatgg gcaacccgag 2160

aataactaca aaaccacacc gcccgtcttg gacagcgatg gcagtttctt cctgtactcc 2220

aaactgactg tggacaaaag ccggtggcag caaggcaatg tgttctcatg ctctgtcatg 2280

cacgaagccc tgcacaacca ttacacccaa aagtcactga gcctgagccc tggaaag 2337

<210> 62

<211> 2322

<212> DNA

<213> Artificial sequence

<220>

<223> F_DS-Cav1-1GS-Fc

<400> 62

atggaacttc tcatcctgaa agccaatgcc attacgacca ttctgacagc cgtaaccttt 60

tgctttgcaa gtggtcagaa tatcactgag gaattctacc agagtacctg tagcgcagtt 120

tctaaggggt acttgtctgc gcttcgcact ggctggtaca cctcggttat taccattgag 180

ctgtccaaca tcaaagagaa caagtgcaac ggcaccgacg ccaaagtgaa actgattaaa 240

caggagctcg acaaatacaa aaatgctgtg acagagttgc agttgctcat gcagtctacc 300

cctgctacga acaaccgagc aaggcgagaa ctaccaagat tcatgaacta caccctgaat 360

aatgcgaaaa agactaacgt gaccctcagc aagaagagaa agaggcgttt tcttgggttt 420

ttgctgggag ttgggagcgc tatagccagc ggagttgctg tgtgtaaggt attgcaccta 480

gaaggggagg tgaataagat caagagtgcc ctgctgtcca caaacaaagc agtagtgtcg 540

cttagcaatg gggtgtccgt tctgaccttc aaggttctag atctcaaaaa ctacatcgat 600

aagcagcttc tgccaatcct caacaagcag tcttgcagta ttagcaatat cgagacggtg 660

atagagtttc aacagaagaa caatcgtctg ctcgaaatta cacgggagtt tagcgtcaac 720

gcaggagtga ctactcctgt aagcacctac atgttaacaa actccgagct gctatctctg 780

atcaatgaca tgccaattac aaacgaccag aaaaagttaa tgtcaaacaa tgtgcagata 840

gtcaggcaac agtcctattc cattatgtgc atcatcaagg aagaagttct ggcctatgtc 900

gtccaacttc ccttatatgg cgtcatagac acgccctgtt ggaaactgca caccagtcct 960

ttgtgcacta caaacactaa ggaggggtct aacatctgtc tgactaggac agatcgcggg 1020

tggtattgcg acaatgctgg ctcagtgagc tttttcccac aagccgaaac atgcaaagtc 1080

cagtcgaatc gggtgttttg tgacacaatg aactcactga ctcttccttc cgaagtgaac 1140

ttgtgcaatg tcgatatctt caatcccaaa tatgactgca agatcatgac aagtaagacc 1200

gatgtcagca gtagcgtcat tacctccctc ggtgctattg tgtcctgtta cggcaagacc 1260

aaatgtactg cttctaacaa aaatcgcggc attattaaga cattcagcaa cggatgtgac 1320

tatgtctcca ataaaggtgt agacacggtg tctgtgggta ataccctcta ctatgtgaat 1380

aagcaggaag gaaagtcact ctatgtgaaa ggagagccga tcatcaactt ctacgatccc 1440

ctggtgtttc ccagtgatga gttcgacgcc tctatcagcc aggtgaatga aaagatcaac 1500

caatccctgg ccttcatacg gaaatcagat gagctgttag gctcaggcta catacccgaa 1560

gcaccgagag atggtcaagc gtatgtgcgg aaggatggag agtgggtcct tctgtcaact 1620

ttcctgggcg gaggaggcag cgacaaaact cacacttgtc cgccttgtcc cgctccagag 1680

ctccttggcg gaccctccgt tttcctgttt cctccgaaac ccaaggatac gctgatgatt 1740

tcacgcacac cagaagtgac atgtgtggtg gtagatgtgt cccatgaaga tccggaggtg 1800

aagtttaact ggtatgtcga tggtgtggag gttcataacg ctaagacgaa accacggggag 1860

gagcagtaca attccaccta ccgtgtagtc tctgtgctga ccgttttgca tcaggattgg 1920

ctgaatggta aagagtataa gtgcaaagtg tccaacaagg ctctgccagc ccctatcgaa 1980

aagaccatca gtaaagccaa aggacagcct agagaacccc aggtctatac gctgccaccc 2040

tctcgggacg agctgaccaa gaatcaggtg tcactgactt gtctggtgaa gggcttctac 2100

cctagcgaca ttgccgtcga atgggagtct aatgggcaac ccgagaataa ctacaaaacc 2160

acaccgcccg tcttggacag cgatggcagt ttcttcctgt actccaaact gactgtggac 2220

aaaagccggt ggcagcaagg caatgtgttc tcatgctctg tcatgcacga agccctgcac 2280

aaccattaca cccaaaagtc actgagcctg agccctggaa ag 2322

<210> 63

<211> 2367

<212> DNA

<213> Artificial sequence

<220>

<223> FH_85-4GS-Fc

<400> 63

atggaactgc tgatacttaa ggctaacgcg ataactacga tcctcaccgc cgtgaccttt 60

tgctttgcct ctggccaaaa cattactgag gaattctacc agtcaacgtg cagtgcagtg 120

agccgaggt atctgtccgc cctgagaacc gggtggtata cttccgtcat taccatcatg 180

ctgtccaaca taaaagagaa taagtgcaac ggcaccgatg ctaaagtgaa actgatcaaa 240

caggaactcg ataaatacaa gaatgcagtt acagagcttc agctcctgat gcagagcact 300

ccggccacca ataatagggc aaggagagaa ttgccacgat ttatgaatta cacactcaac 360

aacgcgaaga aaactaacgt gactctgtcc aagaaacgta agaataactt cttggggttc 420

tgtctgggtg taggtagcgc cattgcttct ggggtggccg tcagcaaagt gcttcacctg 480

gaaggagagg tgaacaagat caagtctgca ctgctgtcta caaacaaagc agtggtgagc 540

ctgtccaacg gagtatccgt tctggtggtc aaagtcctgg atctgaagaa ttatatcgac 600

aaacaactgc tccccattgt gaacaagcag agttgttcaa tcagcaacat agaaactgtg 660

attgagttcc aacagaagaa caataggctg ctcgaaatta ccagagagtt tagcgtcaat 720

gctggtgtca caaccccagt cagcacttac atgctgacta attccgagtt gcttagcctt 780

attaacgaca tgcctatcac caatgaccag aagaagctga tgagtaataa tgtgcagatt 840

gtgcgccagc agagttacag cattatgagt attatcaaag aggaggtatt ggcttatgtg 900

gttcagcttc cgctgtatgg ggtcatcgac acaccttgtt ggaagttgca taccagtccc 960

ctgtgtacga caaacaccaa ggaaggtagt aacatctgct tgacacgtac cgatcggggt 1020

tggtattgcg ataacgccgg gtctgttagt ttctttcctc aagccgagac atgcaaagtc 1080

cagagcaatc gcgtgttctg tgacacgatg aacagctgta ctttgccatc agaggttaat 1140

ctgtgcaata ccgacatctt caaccccaaa tacgactgta agatcatgac cagcaagact 1200

gatgtcagct cctccgttat aacatcactc ggcgctatcg tgtcttgcta tggcaagacc 1260

aagtgtacag cgtccaataa gaatcggggc attatcaaga cattctccaa cggatgtgac 1320

tacgtgagca acaaaggagt ggacaccgtg tcagtcggaa atacactgta ttacgtgaat 1380

aagcaggagg gcaaatctct ttacgtgaag ggcgaaccaa tcatcaactt ctatgatccc 1440

ctcgtctttc cttctgatga gtttgacgcc tctatttctc aggttaacga gaagatcaat 1500

cagtctctgg cctttatacg caaaagcgat gaactcctgg gctcaggcta catacccgaa 1560

gcaccgagag atggtcaagc gtatgtgcgg aaggatggag agtgggtcct tctgtcaact 1620

ttcctgggcg ggggtgggag cggaggcgga ggttcagggg gtggagggtc aggcggagga 1680

ggcagcgaca aaactcacac ttgtccgcct tgtcccgctc cagagctcct tggcggaccc 1740

tccgttttcc tgtttcctcc gaaacccaag gatacgctga tgatttcacg cacaccagaa 1800

gtgacatgtg tggtggtaga tgtgtcccat gaagatccgg aggtgaagtt taactggtat 1860

gtcgatggtg tggaggttca taacgctaag acgaaaccac gggaggagca gtacaattcc 1920

acctaccgtg tagtctctgt gctgaccgtt ttgcatcagg attggctgaa tggtaaagag 1980

tataagtgca aagtgtccaa caaggctctg ccagccccta tcgaaaagac catcagtaaa 2040

gccaaaggac agcctagaga accccaggtc tatacgctgc caccctctcg ggacgagctg 2100

accaagaatc aggtgtcact gacttgtctg gtgaagggct tctaccctag cgacattgcc 2160

gtcgaatggg agtctaatgg gcaacccgag aataactaca aaaccacacc gcccgtcttg 2220

gacagcgatg gcagtttctt cctgtactcc aaactgactg tggacaaaag ccggtggcag 2280

caaggcaatg tgttctcatg ctctgtcatg cacgaagccc tgcacaacca ttacacccaa 2340

aagtcactga gcctgagccc tggaaag 2367

<210> 64

<211> 2337

<212> DNA

<213> Artificial sequence

<220>

<223> FH_85-2GS-Fc

<400> 64

atggaactgc tgatacttaa ggctaacgcg ataactacga tcctcaccgc cgtgaccttt 60

tgctttgcct ctggccaaaa cattactgag gaattctacc agtcaacgtg cagtgcagtg 120

agccgaggt atctgtccgc cctgagaacc gggtggtata cttccgtcat taccatcatg 180

ctgtccaaca taaaagagaa taagtgcaac ggcaccgatg ctaaagtgaa actgatcaaa 240

caggaactcg ataaatacaa gaatgcagtt acagagcttc agctcctgat gcagagcact 300

ccggccacca ataatagggc aaggagagaa ttgccacgat ttatgaatta cacactcaac 360

aacgcgaaga aaactaacgt gactctgtcc aagaaacgta agaataactt cttggggttc 420

tgtctgggtg taggtagcgc cattgcttct ggggtggccg tcagcaaagt gcttcacctg 480

gaaggagagg tgaacaagat caagtctgca ctgctgtcta caaacaaagc agtggtgagc 540

ctgtccaacg gagtatccgt tctggtggtc aaagtcctgg atctgaagaa ttatatcgac 600

aaacaactgc tccccattgt gaacaagcag agttgttcaa tcagcaacat agaaactgtg 660

attgagttcc aacagaagaa caataggctg ctcgaaatta ccagagagtt tagcgtcaat 720

gctggtgtca caaccccagt cagcacttac atgctgacta attccgagtt gcttagcctt 780

attaacgaca tgcctatcac caatgaccag aagaagctga tgagtaataa tgtgcagatt 840

gtgcgccagc agagttacag cattatgagt attatcaaag aggaggtatt ggcttatgtg 900

gttcagcttc cgctgtatgg ggtcatcgac acaccttgtt ggaagttgca taccagtccc 960

ctgtgtacga caaacaccaa ggaaggtagt aacatctgct tgacacgtac cgatcggggt 1020

tggtattgcg ataacgccgg gtctgttagt ttctttcctc aagccgagac atgcaaagtc 1080

cagagcaatc gcgtgttctg tgacacgatg aacagctgta ctttgccatc agaggttaat 1140

ctgtgcaata ccgacatctt caaccccaaa tacgactgta agatcatgac cagcaagact 1200

gatgtcagct cctccgttat aacatcactc ggcgctatcg tgtcttgcta tggcaagacc 1260

aagtgtacag cgtccaataa gaatcggggc attatcaaga cattctccaa cggatgtgac 1320

tacgtgagca acaaaggagt ggacaccgtg tcagtcggaa atacactgta ttacgtgaat 1380

aagcaggagg gcaaatctct ttacgtgaag ggcgaaccaa tcatcaactt ctatgatccc 1440

ctcgtctttc cttctgatga gtttgacgcc tctatttctc aggttaacga gaagatcaat 1500

cagtctctgg cctttatacg caaaagcgat gaactcctgg gctcaggcta catacccgaa 1560

gcaccgagag atggtcaagc gtatgtgcgg aaggatggag agtgggtcct tctgtcaact 1620

ttcctggggg gtggagggtc aggcggagga ggcagcgaca aaactcacac ttgtccgcct 1680

tgtcccgctc cagagctcct tggcggaccc tccgttttcc tgtttcctcc gaaacccaag 1740

gatacgctga tgatttcacg cacaccagaa gtgacatgtg tggtggtaga tgtgtcccat 1800

gaagatccgg aggtgaagtt taactggtat gtcgatggtg tggaggttca taacgctaag 1860

acgaaaccac gggaggagca gtacaattcc acctaccgtg tagtctctgt gctgaccgtt 1920

ttgcatcagg attggctgaa tggtaaagag tataagtgca aagtgtccaa caaggctctg 1980

ccagccccta tcgaaaagac catcagtaaa gccaaaggac agcctagaga accccaggtc 2040

tatacgctgc caccctctcg ggacgagctg accaagaatc aggtgtcact gacttgtctg 2100

gtgaagggct tctaccctag cgacattgcc gtcgaatggg agtctaatgg gcaacccgag 2160

aataactaca aaaccacacc gcccgtcttg gacagcgatg gcagtttctt cctgtactcc 2220

aaactgactg tggacaaaag ccggtggcag caaggcaatg tgttctcatg ctctgtcatg 2280

cacgaagccc tgcacaacca ttacacccaa aagtcactga gcctgagccc tggaaag 2337

<210> 65

<211> 2322

<212> DNA

<213> Artificial sequence

<220>

<223> FH_85-1GS-Fc

<400> 65

atggaactgc tgatacttaa ggctaacgcg ataactacga tcctcaccgc cgtgaccttt 60

tgctttgcct ctggccaaaa cattactgag gaattctacc agtcaacgtg cagtgcagtg 120

agccgaggt atctgtccgc cctgagaacc gggtggtata cttccgtcat taccatcatg 180

ctgtccaaca taaaagagaa taagtgcaac ggcaccgatg ctaaagtgaa actgatcaaa 240

caggaactcg ataaatacaa gaatgcagtt acagagcttc agctcctgat gcagagcact 300

ccggccacca ataatagggc aaggagagaa ttgccacgat ttatgaatta cacactcaac 360

aacgcgaaga aaactaacgt gactctgtcc aagaaacgta agaataactt cttggggttc 420

tgtctgggtg taggtagcgc cattgcttct ggggtggccg tcagcaaagt gcttcacctg 480

gaaggagagg tgaacaagat caagtctgca ctgctgtcta caaacaaagc agtggtgagc 540

ctgtccaacg gagtatccgt tctggtggtc aaagtcctgg atctgaagaa ttatatcgac 600

aaacaactgc tccccattgt gaacaagcag agttgttcaa tcagcaacat agaaactgtg 660

attgagttcc aacagaagaa caataggctg ctcgaaatta ccagagagtt tagcgtcaat 720

gctggtgtca caaccccagt cagcacttac atgctgacta attccgagtt gcttagcctt 780

attaacgaca tgcctatcac caatgaccag aagaagctga tgagtaataa tgtgcagatt 840

gtgcgccagc agagttacag cattatgagt attatcaaag aggaggtatt ggcttatgtg 900

gttcagcttc cgctgtatgg ggtcatcgac acaccttgtt ggaagttgca taccagtccc 960

ctgtgtacga caaacaccaa ggaaggtagt aacatctgct tgacacgtac cgatcggggt 1020

tggtattgcg ataacgccgg gtctgttagt ttctttcctc aagccgagac atgcaaagtc 1080

cagagcaatc gcgtgttctg tgacacgatg aacagctgta ctttgccatc agaggttaat 1140

ctgtgcaata ccgacatctt caaccccaaa tacgactgta agatcatgac cagcaagact 1200

gatgtcagct cctccgttat aacatcactc ggcgctatcg tgtcttgcta tggcaagacc 1260

aagtgtacag cgtccaataa gaatcggggc attatcaaga cattctccaa cggatgtgac 1320

tacgtgagca acaaaggagt ggacaccgtg tcagtcggaa atacactgta ttacgtgaat 1380

aagcaggagg gcaaatctct ttacgtgaag ggcgaaccaa tcatcaactt ctatgatccc 1440

ctcgtctttc cttctgatga gtttgacgcc tctatttctc aggttaacga gaagatcaat 1500

cagtctctgg cctttatacg caaaagcgat gaactcctgg gctcaggcta catacccgaa 1560

gcaccgagag atggtcaagc gtatgtgcgg aaggatggag agtgggtcct tctgtcaact 1620

ttcctgggcg gaggaggcag cgacaaaact cacacttgtc cgccttgtcc cgctccagag 1680

ctccttggcg gaccctccgt tttcctgttt cctccgaaac ccaaggatac gctgatgatt 1740

tcacgcacac cagaagtgac atgtgtggtg gtagatgtgt cccatgaaga tccggaggtg 1800

aagtttaact ggtatgtcga tggtgtggag gttcataacg ctaagacgaa accacggggag 1860

gagcagtaca attccaccta ccgtgtagtc tctgtgctga ccgttttgca tcaggattgg 1920

ctgaatggta aagagtataa gtgcaaagtg tccaacaagg ctctgccagc ccctatcgaa 1980

aagaccatca gtaaagccaa aggacagcct agagaacccc aggtctatac gctgccaccc 2040

tctcgggacg agctgaccaa gaatcaggtg tcactgactt gtctggtgaa gggcttctac 2100

cctagcgaca ttgccgtcga atgggagtct aatgggcaac ccgagaataa ctacaaaacc 2160

acaccgcccg tcttggacag cgatggcagt ttcttcctgt actccaaact gactgtggac 2220

aaaagccggt ggcagcaagg caatgtgttc tcatgctctg tcatgcacga agccctgcac 2280

aaccattaca cccaaaagtc actgagcctg agccctggaa ag 2322

<210> 66

<211> 1713

<212> DNA

<213> Artificial sequence

<220>

<223> F_DS-Cav1-CC02

<400> 66

atggaacttc tcatcctgaa agccaatgcc attacgacca ttctgacagc cgtaaccttt 60

tgctttgcaa gtggtcagaa tatcactgag gaattctacc agagtacctg tagcgcagtt 120

tctaaggggt acttgtctgc gcttcgcact ggctggtaca cctcggttat taccattgag 180

ctgtccaaca tcaaagagaa caagtgcaac ggcaccgacg ccaaagtgaa actgattaaa 240

caggagctcg acaaatacaa aaatgctgtg acagagttgc agttgctcat gcagtctacc 300

cctgctacga acaaccgagc aaggcgagaa ctaccaagat tcatgaacta caccctgaat 360

aatgcgaaaa agactaacgt gaccctcagc aagaagagaa agaggcgttt tcttgggttt 420

ttgctgggag ttgggagcgc tatagccagc ggagttgctg tgtgtaaggt attgcaccta 480

gaaggggagg tgaataagat caagagtgcc ctgctgtcca caaacaaagc agtagtgtcg 540

cttagcaatg gggtgtccgt tctgaccttc aaggttctag atctcaaaaa ctacatcgat 600

aagcagcttc tgccaatcct caacaagcag tcttgcagta ttagcaatat cgagacggtg 660

atagagtttc aacagaagaa caatcgtctg ctcgaaatta cacgggagtt tagcgtcaac 720

gcaggagtga ctactcctgt aagcacctac atgttaacaa actccgagct gctatctctg 780

atcaatgaca tgccaattac aaacgaccag aaaaagttaa tgtcaaacaa tgtgcagata 840

gtcaggcaac agtcctattc cattatgtgc atcatcaagg aagaagttct ggcctatgtc 900

gtccaacttc ccttatatgg cgtcatagac acgccctgtt ggaaactgca caccagtcct 960

ttgtgcacta caaacactaa ggaggggtct aacatctgtc tgactaggac agatcgcggg 1020

tggtattgcg acaatgctgg ctcagtgagc tttttcccac aagccgaaac atgcaaagtc 1080

cagtcgaatc gggtgttttg tgacacaatg aactcactga ctcttccttc cgaagtgaac 1140

ttgtgcaatg tcgatatctt caatcccaaa tatgactgca agatcatgac aagtaagacc 1200

gatgtcagca gtagcgtcat tacctccctc ggtgctattg tgtcctgtta cggcaagacc 1260

aaatgtactg cttctaacaa aaatcgcggc attattaaga cattcagcaa cggatgtgac 1320

tatgtctcca ataaaggtgt agacacggtg tctgtgggta ataccctcta ctatgtgaat 1380

aagcaggaag gaaagtcact ctatgtgaaa ggagagccga tcatcaactt ctacgatccc 1440

ctggtgtttc ccagtgatga gttcgacgcc tctatcagcc aggtgaatga aaagatcaac 1500

caatccctgg ccttcatacg gaaatcagat gagctgttag ggtctggcta catccctgaa 1560

gcacccagag atggtcaggc ctatgtgagg aaagatggcg aatgggtcct gctgtccacc 1620

ttcctgatag cgttggccct ggagaaaatc gcccttgcac tggagaagat tgccctcgcc 1680

ttggagaagg actacaagga cgatgacgac aaa 1713

<210> 67

<211> 1713

<212> DNA

<213> Artificial sequence

<220>

<223> F_DS-Cav1-CC03

<400> 67

atggaacttc tcatcctgaa agccaatgcc attacgacca ttctgacagc cgtaaccttt 60

tgctttgcaa gtggtcagaa tatcactgag gaattctacc agagtacctg tagcgcagtt 120

tctaaggggt acttgtctgc gcttcgcact ggctggtaca cctcggttat taccattgag 180

ctgtccaaca tcaaagagaa caagtgcaac ggcaccgacg ccaaagtgaa actgattaaa 240

caggagctcg acaaatacaa aaatgctgtg acagagttgc agttgctcat gcagtctacc 300

cctgctacga acaaccgagc aaggcgagaa ctaccaagat tcatgaacta caccctgaat 360

aatgcgaaaa agactaacgt gaccctcagc aagaagagaa agaggcgttt tcttgggttt 420

ttgctgggag ttgggagcgc tatagccagc ggagttgctg tgtgtaaggt attgcaccta 480

gaaggggagg tgaataagat caagagtgcc ctgctgtcca caaacaaagc agtagtgtcg 540

cttagcaatg gggtgtccgt tctgaccttc aaggttctag atctcaaaaa ctacatcgat 600

aagcagcttc tgccaatcct caacaagcag tcttgcagta ttagcaatat cgagacggtg 660

atagagtttc aacagaagaa caatcgtctg ctcgaaatta cacgggagtt tagcgtcaac 720

gcaggagtga ctactcctgt aagcacctac atgttaacaa actccgagct gctatctctg 780

atcaatgaca tgccaattac aaacgaccag aaaaagttaa tgtcaaacaa tgtgcagata 840

gtcaggcaac agtcctattc cattatgtgc atcatcaagg aagaagttct ggcctatgtc 900

gtccaacttc ccttatatgg cgtcatagac acgccctgtt ggaaactgca caccagtcct 960

ttgtgcacta caaacactaa ggaggggtct aacatctgtc tgactaggac agatcgcggg 1020

tggtattgcg acaatgctgg ctcagtgagc tttttcccac aagccgaaac atgcaaagtc 1080

cagtcgaatc gggtgttttg tgacacaatg aactcactga ctcttccttc cgaagtgaac 1140

ttgtgcaatg tcgatatctt caatcccaaa tatgactgca agatcatgac aagtaagacc 1200

gatgtcagca gtagcgtcat tacctccctc ggtgctattg tgtcctgtta cggcaagacc 1260

aaatgtactg cttctaacaa aaatcgcggc attattaaga cattcagcaa cggatgtgac 1320

tatgtctcca ataaaggtgt agacacggtg tctgtgggta ataccctcta ctatgtgaat 1380

aagcaggaag gaaagtcact ctatgtgaaa ggagagccga tcatcaactt ctacgatccc 1440

ctggtgtttc ccagtgatga gttcgacgcc tctatcagcc aggtgaatga aaagatcaac 1500

caatccctgg ccttcatacg gaaatcagat gagctgttag gatctggcta cattcccgaa 1560

gctcctagag atggccaggc ctatgtgagg aaagatgggg agtgggtgtt gctgagcacc 1620

ttcctcatca aactggagct ggagaagatt aagctggaac tggagaaaat caagctcgaa 1680

cttgagaagg actacaagga cgatgacgac aaa 1713

<210> 68

<211> 1713

<212> DNA

<213> Artificial sequence

<220>

<223> F_DS-Cav1-CC07

<400> 68

atggaacttc tcatcctgaa agccaatgcc attacgacca ttctgacagc cgtaaccttt 60

tgctttgcaa gtggtcagaa tatcactgag gaattctacc agagtacctg tagcgcagtt 120

tctaaggggt acttgtctgc gcttcgcact ggctggtaca cctcggttat taccattgag 180

ctgtccaaca tcaaagagaa caagtgcaac ggcaccgacg ccaaagtgaa actgattaaa 240

caggagctcg acaaatacaa aaatgctgtg acagagttgc agttgctcat gcagtctacc 300

cctgctacga acaaccgagc aaggcgagaa ctaccaagat tcatgaacta caccctgaat 360

aatgcgaaaa agactaacgt gaccctcagc aagaagagaa agaggcgttt tcttgggttt 420

ttgctgggag ttgggagcgc tatagccagc ggagttgctg tgtgtaaggt attgcaccta 480

gaaggggagg tgaataagat caagagtgcc ctgctgtcca caaacaaagc agtagtgtcg 540

cttagcaatg gggtgtccgt tctgaccttc aaggttctag atctcaaaaa ctacatcgat 600

aagcagcttc tgccaatcct caacaagcag tcttgcagta ttagcaatat cgagacggtg 660

atagagtttc aacagaagaa caatcgtctg ctcgaaatta cacgggagtt tagcgtcaac 720

gcaggagtga ctactcctgt aagcacctac atgttaacaa actccgagct gctatctctg 780

atcaatgaca tgccaattac aaacgaccag aaaaagttaa tgtcaaacaa tgtgcagata 840

gtcaggcaac agtcctattc cattatgtgc atcatcaagg aagaagttct ggcctatgtc 900

gtccaacttc ccttatatgg cgtcatagac acgccctgtt ggaaactgca caccagtcct 960

ttgtgcacta caaacactaa ggaggggtct aacatctgtc tgactaggac agatcgcggg 1020

tggtattgcg acaatgctgg ctcagtgagc tttttcccac aagccgaaac atgcaaagtc 1080

cagtcgaatc gggtgttttg tgacacaatg aactcactga ctcttccttc cgaagtgaac 1140

ttgtgcaatg tcgatatctt caatcccaaa tatgactgca agatcatgac aagtaagacc 1200

gatgtcagca gtagcgtcat tacctccctc ggtgctattg tgtcctgtta cggcaagacc 1260

aaatgtactg cttctaacaa aaatcgcggc attattaaga cattcagcaa cggatgtgac 1320

tatgtctcca ataaaggtgt agacacggtg tctgtgggta ataccctcta ctatgtgaat 1380

aagcaggaag gaaagtcact ctatgtgaaa ggagagccga tcatcaactt ctacgatccc 1440

ctggtgtttc ccagtgatga gttcgacgcc tctatcagcc aggtgaatga aaagatcaac 1500

caatccctgg ccttcatacg gaaatcagat gagctgttag gttccgggta catacccgaa 1560

gctcctagag atggacaggc ctatgtgagg aaagatggcg aatgggtctt actgagcacc 1620

ttcttgctcc atctgcacat tgagaagcta cacctccata tcgagaaact gcatcttcac 1680

atcgagaagg actacaagga cgatgacgac aaa 1713

<210> 69

<211> 1713

<212> DNA

<213> Artificial sequence

<220>

<223> F_DS-Cav1-CC08

<400> 69

atggaacttc tcatcctgaa agccaatgcc attacgacca ttctgacagc cgtaaccttt 60

tgctttgcaa gtggtcagaa tatcactgag gaattctacc agagtacctg tagcgcagtt 120

tctaaggggt acttgtctgc gcttcgcact ggctggtaca cctcggttat taccattgag 180

ctgtccaaca tcaaagagaa caagtgcaac ggcaccgacg ccaaagtgaa actgattaaa 240

caggagctcg acaaatacaa aaatgctgtg acagagttgc agttgctcat gcagtctacc 300

cctgctacga acaaccgagc aaggcgagaa ctaccaagat tcatgaacta caccctgaat 360

aatgcgaaaa agactaacgt gaccctcagc aagaagagaa agaggcgttt tcttgggttt 420

ttgctgggag ttgggagcgc tatagccagc ggagttgctg tgtgtaaggt attgcaccta 480

gaaggggagg tgaataagat caagagtgcc ctgctgtcca caaacaaagc agtagtgtcg 540

cttagcaatg gggtgtccgt tctgaccttc aaggttctag atctcaaaaa ctacatcgat 600

aagcagcttc tgccaatcct caacaagcag tcttgcagta ttagcaatat cgagacggtg 660

atagagtttc aacagaagaa caatcgtctg ctcgaaatta cacgggagtt tagcgtcaac 720

gcaggagtga ctactcctgt aagcacctac atgttaacaa actccgagct gctatctctg 780

atcaatgaca tgccaattac aaacgaccag aaaaagttaa tgtcaaacaa tgtgcagata 840

gtcaggcaac agtcctattc cattatgtgc atcatcaagg aagaagttct ggcctatgtc 900

gtccaacttc ccttatatgg cgtcatagac acgccctgtt ggaaactgca caccagtcct 960

ttgtgcacta caaacactaa ggaggggtct aacatctgtc tgactaggac agatcgcggg 1020

tggtattgcg acaatgctgg ctcagtgagc tttttcccac aagccgaaac atgcaaagtc 1080

cagtcgaatc gggtgttttg tgacacaatg aactcactga ctcttccttc cgaagtgaac 1140

ttgtgcaatg tcgatatctt caatcccaaa tatgactgca agatcatgac aagtaagacc 1200

gatgtcagca gtagcgtcat tacctccctc ggtgctattg tgtcctgtta cggcaagacc 1260

aaatgtactg cttctaacaa aaatcgcggc attattaaga cattcagcaa cggatgtgac 1320

tatgtctcca ataaaggtgt agacacggtg tctgtgggta ataccctcta ctatgtgaat 1380

aagcaggaag gaaagtcact ctatgtgaaa ggagagccga tcatcaactt ctacgatccc 1440

ctggtgtttc ccagtgatga gttcgacgcc tctatcagcc aggtgaatga aaagatcaac 1500

caatccctgg ccttcatacg gaaatcagat gagctgttag gaagcggcta cattcccgaa 1560

gctcctagag atggtcaggc ctatgtgagg aaagatgggg aatgggtctt actgtccacc 1620

ttccttctca agttggagat cccatctg aagctagaga tccatcacct caaactggag 1680

atacaccatg actacaagga cgatgacgac aaa 1713

<210> 70

<211> 1713

<212> DNA

<213> Artificial sequence

<220>

<223> FH_82-CC07

<400> 70

atggaactgc tgatacttaa ggctaacgcg ataactacga tcctcaccgc cgtgaccttt 60

tgctttgcct ctggccaaaa cattactgag gaattctacc agtcaacgtg cagtgcagtg 120

agccgaggt atctgtccgc cctgagaacc gggtggtata cttccgtcat taccatcgag 180

ctgtccaaca taaaagagaa taagtgcaac ggcaccgatg ctaaagtgaa actgatcaaa 240

caggaactcg ataaatacaa gaatgcagtt acagagcttc agctcctgat gcagagcact 300

ccggccacca ataatagggc aaggagagaa ttgccacgat ttatgaatta cacactcaac 360

aacgcgaaga aaactaacgt gactctgtcc aagaaacgta agaataactt cttggggttc 420

tgtctgggtg taggtagcgc cattgcttct ggggtggccg tcagcaaagt gcttcacctg 480

gaaggagagg tgaacaagat caagtctgca ctgctgtcta caaacaaagc agtggtgagc 540

ctgtccaacg gagtatccgt tctggtggtc aaagtcctgg atctgaagaa ttatatcgac 600

aaacaactgc tccccattgt gaacaagcag agttgttcaa tcagcaacat agaaactgtg 660

attgagttcc aacagaagaa caataggctg ctcgaaatta ccagagagtt tagcgtcaat 720

gctggtgtca caaccccagt cagcacttac atgctgacta attccgagtt gcttagcctt 780

attaacgaca tgcctatcac caatgaccag aagaagctga tgagtaataa tgtgcagatt 840

gtgcgccagc agagttacag cattatgagt attatcaaag aggaggtatt ggcttatgtg 900

gttcagcttc cgctgtatgg ggtcatcgac acaccttgtt ggaagttgca taccagtccc 960

ctgtgtacga caaacaccaa ggaaggtagt aacatctgct tgacacgtac cgatcggggt 1020

tggtattgcg ataacgccgg gtctgttagt ttctttcctc aagccgagac atgcaaagtc 1080

cagagcaatc gcgtgttctg tgacacgatg aacagctgta ctttgccatc agaggttaat 1140

ctgtgcaata ccgacatctt caaccccaaa tacgactgta agatcatgac cagcaagact 1200

gatgtcagct cctccgttat aacatcactc ggcgctatcg tgtcttgcta tggcaagacc 1260

aagtgtacag cgtccaataa gaatcggggc attatcaaga cattctccaa cggatgtgac 1320

tacgtgagca acaaaggagt ggacaccgtg tcagtcggaa atacactgta ttacgtgaat 1380

aagcaggagg gcaaatctct ttacgtgaag ggcgaaccaa tcatcaactt ctatgatccc 1440

ctcgtctttc cttctgatga gtttgacgcc tctatttctc aggttaacga gaagatcaat 1500

cagtctctgg cctttatacg caaaagcgat gaactcctgg gatcaggcta cattcccgaa 1560

gctccaaggg acggacaagc atacgtacga aaagatggcg aatgggtctt actgagcacc 1620

ttcttgctcc atctgcacat tgagaagcta cacctccata tcgagaaact gcatcttcac 1680

atcgagaagg actacaagga cgatgacgac aaa 1713

<210> 71

<211> 1713

<212> DNA

<213> Artificial sequence

<220>

<223> FH_85-CC07

<400> 71

atggaactgc tgatacttaa ggctaacgcg ataactacga tcctcaccgc cgtgaccttt 60

tgctttgcct ctggccaaaa cattactgag gaattctacc agtcaacgtg cagtgcagtg 120

agccgaggt atctgtccgc cctgagaacc gggtggtata cttccgtcat taccatcatg 180

ctgtccaaca taaaagagaa taagtgcaac ggcaccgatg ctaaagtgaa actgatcaaa 240

caggaactcg ataaatacaa gaatgcagtt acagagcttc agctcctgat gcagagcact 300

ccggccacca ataatagggc aaggagagaa ttgccacgat ttatgaatta cacactcaac 360

aacgcgaaga aaactaacgt gactctgtcc aagaaacgta agaataactt cttggggttc 420

tgtctgggtg taggtagcgc cattgcttct ggggtggccg tcagcaaagt gcttcacctg 480

gaaggagagg tgaacaagat caagtctgca ctgctgtcta caaacaaagc agtggtgagc 540

ctgtccaacg gagtatccgt tctggtggtc aaagtcctgg atctgaagaa ttatatcgac 600

aaacaactgc tccccattgt gaacaagcag agttgttcaa tcagcaacat agaaactgtg 660

attgagttcc aacagaagaa caataggctg ctcgaaatta ccagagagtt tagcgtcaat 720

gctggtgtca caaccccagt cagcacttac atgctgacta attccgagtt gcttagcctt 780

attaacgaca tgcctatcac caatgaccag aagaagctga tgagtaataa tgtgcagatt 840

gtgcgccagc agagttacag cattatgagt attatcaaag aggaggtatt ggcttatgtg 900

gttcagcttc cgctgtatgg ggtcatcgac acaccttgtt ggaagttgca taccagtccc 960

ctgtgtacga caaacaccaa ggaaggtagt aacatctgct tgacacgtac cgatcggggt 1020

tggtattgcg ataacgccgg gtctgttagt ttctttcctc aagccgagac atgcaaagtc 1080

cagagcaatc gcgtgttctg tgacacgatg aacagctgta ctttgccatc agaggttaat 1140

ctgtgcaata ccgacatctt caaccccaaa tacgactgta agatcatgac cagcaagact 1200

gatgtcagct cctccgttat aacatcactc ggcgctatcg tgtcttgcta tggcaagacc 1260

aagtgtacag cgtccaataa gaatcggggc attatcaaga cattctccaa cggatgtgac 1320

tacgtgagca acaaaggagt ggacaccgtg tcagtcggaa atacactgta ttacgtgaat 1380

aagcaggagg gcaaatctct ttacgtgaag ggcgaaccaa tcatcaactt ctatgatccc 1440

ctcgtctttc cttctgatga gtttgacgcc tctatttctc aggttaacga gaagatcaat 1500

cagtctctgg cctttatacg caaaagcgat gaactcctgg gatcaggcta cattcccgaa 1560

gctccaaggg acggacaagc atacgtacga aaagatggcg aatgggtctt actgagcacc 1620

ttcttgctcc atctgcacat tgagaagcta cacctccata tcgagaaact gcatcttcac 1680

atcgagaagg actacaagga cgatgacgac aaa 1713

<210> 72

<211> 2184

<212> DNA

<213> Artificial sequence

<220>

<223> F_DC-Cav1-HBcCN

<400> 72

atggaacttc tcatcctgaa agccaatgcc attacgacca ttctgacagc cgtaaccttt 60

tgctttgcaa gtggtcagaa tatcactgag gaattctacc agagtacctg tagcgcagtt 120

tctaaggggt acttgtctgc gcttcgcact ggctggtaca cctcggttat taccattgag 180

ctgtccaaca tcaaagagaa caagtgcaac ggcaccgacg ccaaagtgaa actgattaaa 240

caggagctcg acaaatacaa aaatgctgtg acagagttgc agttgctcat gcagtctacc 300

cctgctacga acaaccgagc aaggcgagaa ctaccaagat tcatgaacta caccctgaat 360

aatgcgaaaa agactaacgt gaccctcagc aagaagagaa agaggcgttt tcttgggttt 420

ttgctgggag ttgggagcgc tatagccagc ggagttgctg tgtgtaaggt attgcaccta 480

gaaggggagg tgaataagat caagagtgcc ctgctgtcca caaacaaagc agtagtgtcg 540

cttagcaatg gggtgtccgt tctgaccttc aaggttctag atctcaaaaa ctacatcgat 600

aagcagcttc tgccaatcct caacaagcag tcttgcagta ttagcaatat cgagacggtg 660

atagagtttc aacagaagaa caatcgtctg ctcgaaatta cacgggagtt tagcgtcaac 720

gcaggagtga ctactcctgt aagcacctac atgttaacaa actccgagct gctatctctg 780

atcaatgaca tgccaattac aaacgaccag aaaaagttaa tgtcaaacaa tgtgcagata 840

gtcaggcaac agtcctattc cattatgtgc atcatcaagg aagaagttct ggcctatgtc 900

gtccaacttc ccttatatgg cgtcatagac acgccctgtt ggaaactgca caccagtcct 960

ttgtgcacta caaacactaa ggaggggtct aacatctgtc tgactaggac agatcgcggg 1020

tggtattgcg acaatgctgg ctcagtgagc tttttcccac aagccgaaac atgcaaagtc 1080

cagtcgaatc gggtgttttg tgacacaatg aactcactga ctcttccttc cgaagtgaac 1140

ttgtgcaatg tcgatatctt caatcccaaa tatgactgca agatcatgac aagtaagacc 1200

gatgtcagca gtagcgtcat tacctccctc ggtgctattg tgtcctgtta cggcaagacc 1260

aaatgtactg cttctaacaa aaatcgcggc attattaaga cattcagcaa cggatgtgac 1320

tatgtctcca ataaaggtgt agacacggtg tctgtgggta ataccctcta ctatgtgaat 1380

aagcaggaag gaaagtcact ctatgtgaaa ggagagccga tcatcaactt ctacgatccc 1440

ctggtgtttc ccagtgatga gttcgacgcc tctatcagcc aggtgaatga aaagatcaac 1500

caatccctgg ccttcatacg gaaatcagat gagctgttag gctcaggcta catacccgaa 1560

gcaccgagag atggtcaagc gtatgtgcgg aaggatggag agtgggtcct tctgtcaact 1620

ttcctgggcg ggggtgggag cggaggcgga ggttcagggg gtggagggtc aggcggagga 1680

ggcagcgctt ccagggagtt agtggtctcc tacgtcaacg tgaacatggg ccttaagatc 1740

cgtcagttgc tgtggttcca catttccgcc ctaacctttg gcagagagac tgtgctggag 1800

tacctggtgt catttggcgt gtggattcgc actcctccag cagcaagacc cccaaatgcg 1860

cctatcctgt ccacgttacc agagaccacc gttgttgggg gaggtggttc tggagggggc 1920

ggatctggcg gaggtggcag tggggggcggt ggaagtggcg ctagcgtcga gttgctcagc 1980

ttccttccct cggacttctt tccgtccatc agggatctgc tggacacagc cagtgctctc 2040

tatcgggaag cactggaatc tcccgaacac agctctcctc accatacagc ccttcgacaa 2100

gccatactct gctggggcga actgatgaat ctcgccacat gggtagggtc aggggggagc 2160

gactacaagg acgatgacga caaa 2184

<210> 73

<211> 2184

<212> DNA

<213> Artificial sequence

<220>

<223> FH_85-HBcCN

<400> 73

atggaactgc tgatacttaa ggctaacgcg ataactacga tcctcaccgc cgtgaccttt 60

tgctttgcct ctggccaaaa cattactgag gaattctacc agtcaacgtg cagtgcagtg 120

agccgaggt atctgtccgc cctgagaacc gggtggtata cttccgtcat taccatcatg 180

ctgtccaaca taaaagagaa taagtgcaac ggcaccgatg ctaaagtgaa actgatcaaa 240

caggaactcg ataaatacaa gaatgcagtt acagagcttc agctcctgat gcagagcact 300

ccggccacca ataatagggc aaggagagaa ttgccacgat ttatgaatta cacactcaac 360

aacgcgaaga aaactaacgt gactctgtcc aagaaacgta agaataactt cttggggttc 420

tgtctgggtg taggtagcgc cattgcttct ggggtggccg tcagcaaagt gcttcacctg 480

gaaggagagg tgaacaagat caagtctgca ctgctgtcta caaacaaagc agtggtgagc 540

ctgtccaacg gagtatccgt tctggtggtc aaagtcctgg atctgaagaa ttatatcgac 600

aaacaactgc tccccattgt gaacaagcag agttgttcaa tcagcaacat agaaactgtg 660

attgagttcc aacagaagaa caataggctg ctcgaaatta ccagagagtt tagcgtcaat 720

gctggtgtca caaccccagt cagcacttac atgctgacta attccgagtt gcttagcctt 780

attaacgaca tgcctatcac caatgaccag aagaagctga tgagtaataa tgtgcagatt 840

gtgcgccagc agagttacag cattatgagt attatcaaag aggaggtatt ggcttatgtg 900

gttcagcttc cgctgtatgg ggtcatcgac acaccttgtt ggaagttgca taccagtccc 960

ctgtgtacga caaacaccaa ggaaggtagt aacatctgct tgacacgtac cgatcggggt 1020

tggtattgcg ataacgccgg gtctgttagt ttctttcctc aagccgagac atgcaaagtc 1080

cagagcaatc gcgtgttctg tgacacgatg aacagctgta ctttgccatc agaggttaat 1140

ctgtgcaata ccgacatctt caaccccaaa tacgactgta agatcatgac cagcaagact 1200

gatgtcagct cctccgttat aacatcactc ggcgctatcg tgtcttgcta tggcaagacc 1260

aagtgtacag cgtccaataa gaatcggggc attatcaaga cattctccaa cggatgtgac 1320

tacgtgagca acaaaggagt ggacaccgtg tcagtcggaa atacactgta ttacgtgaat 1380

aagcaggagg gcaaatctct ttacgtgaag ggcgaaccaa tcatcaactt ctatgatccc 1440

ctcgtctttc cttctgatga gtttgacgcc tctatttctc aggttaacga gaagatcaat 1500

cagtctctgg cctttatacg caaaagcgat gaactcctgg gatcaggcta cattcccgaa 1560

gctccaaggg acggacaagc gtatgtgcgg aaggatggag agtgggtcct tctgtcaact 1620

ttcctgggcg ggggtgggag cggaggcgga ggttcagggg gtggagggtc aggcggagga 1680

ggcagcgctt ccagggagtt agtggtctcc tacgtcaacg tgaacatggg ccttaagatc 1740

cgtcagttgc tgtggttcca catttccgcc ctaacctttg gcagagagac tgtgctggag 1800

tacctggtgt catttggcgt gtggattcgc actcctccag cagcaagacc cccaaatgcg 1860

cctatcctgt ccacgttacc agagaccacc gttgttgggg gaggtggttc tggagggggc 1920

ggatctggcg gaggtggcag tggggggcggt ggaagtggcg ctagcgtcga gttgctcagc 1980

ttccttccct cggacttctt tccgtccatc agggatctgc tggacacagc cagtgctctc 2040

tatcgggaag cactggaatc tcccgaacac agctctcctc accatacagc ccttcgacaa 2100

gccatactct gctggggcga actgatgaat ctcgccacat gggtagggtc aggggggagc 2160

gactacaagg acgatgacga caaa 2184

<210> 74

<211> 1722

<212> DNA

<213> Artificial sequence

<220>

<223> RSV_F

<400> 74

atggagttgc taatcctcaa agcaaatgca attaccacaa tcctcactgc agtcacattt 60

tgttttgctt ctggtcaaaa catcactgaa gaattttatc aatcaacatg cagtgcagtt 120

agcaaaggct atcttagtgc tctgagaact ggttggtata ccagtgttat aactatagaa 180

ttaagtaata tcaaggaaaa taagtgtaat ggaacagatg ctaaggtaaa attgataaaa 240

caagaattag ataaatataa aaatgctgta acagaattgc agttgctcat gcaaagcaca 300

ccaccaacaa acaatcgagc cagaagagaa ctaccaaggt ttatgaatta tacactcaac 360

aatgccaaaa aaaccaatgt aacattaagc aagaaaagga aaagaagatt tcttggtttt 420

ttgttaggtg ttggatctgc aatcgccagt ggcgttgctg tatctaaggt cctgcaccta 480

gaaggggaag tgaacaagat caaaagtgct ctactatcca caaacaaggc tgtagtcagc 540

ttatcaaatg gagttagtgt cttaaccagc aaagtgttag acctcaaaaa ctatatagat 600

aaacaattgt tacctattgt gaacaagcaa agctgcagca tatcaaatat agaaactgtg 660

atagagttcc aacaaaagaa caacagacta ctagagatta ccagggaatt tagtgttaat 720

gcaggtgtaa ctacacctgt aagcacttac atgttaacta atagtgaatt attgtcatta 780

atcaatgata tgcctataac aaatgatcag aaaaagttaa tgtccaacaa tgttcaaata 840

gttagacagc aaagttactc tatcatgtcc ataataaaag aggaagtctt agcatatgta 900

gtacaattac cactatatgg tgttatagat acaccctgtt ggaaactaca cacatcccct 960

ctatgtacaa ccaacacaaa agaagggtcc aacatctgtt taacaagaac tgacagagga 1020

tggtactgtg acaatgcagg atcagtatct ttcttcccac aagctgaaac atgtaaagtt 1080

caatcaaatc gagtattttg tgacacaatg aacagtttaa cattaccaag tgaaataaat 1140

ctctgcaatg ttgacatatt caaccccaaa tatgattgta aaattatgac ttcaaaaaca 1200

gatgtaagca gctccgttat cacatctcta ggagccattg tgtcatgcta tggcaaaact 1260

aaatgtacag catccaataa aaatcgtgga atcataaaga cattttctaa cgggtgcgat 1320

tatgtatcaa ataaagggat ggacactgtg tctgtaggta acacattata ttatgtaaat 1380

aagcaagaag gtaaaagtct ctatgtaaaa ggtgaaccaa taataaattt ctatgaccca 1440

ttagtattcc cctctgatga atttgatgca tcaatatctc aagtcaacga gaagattaac 1500

cagagcctag catttattcg taaatccgat gaattattac ataatgtaaa tgctggtaaa 1560

tccaccacaa atatcatgat aactactata attatagtga ttatagtaat attgttatca 1620

ttaattgctg ttggactgct cttatactgt aaggccagaa gcacaccagt cacactaagc 1680

aaagatcaac tgagtggtat aaataatatt gcatttagta ac 1722

<210> 75

<211> 20

<212>PRT

<213> Artificial sequence

<220>

<223> GS linker

<400> 75

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

1 5 10 15

Gly Gly Gly Ser

20

<---

Claims (50)

1. A respiratory syncytial virus (RSV) mutant F protein having a mutation, wherein the mutation is a substitution of a leucine corresponding to leucine at position 141 of amino acid sequence SEQ ID NO: 1, or a leucine corresponding to leucine at position 142, with a cysteine, and a substitution of leucine corresponding to leucine at position 373 to cysteine, followed by the formation of a disulfide bond between the cysteines. 2. The mutant RSV F protein of claim 1, wherein the mutant RSV F protein is derived from RSV subtype A or RSV subtype B. 3. The RSV F mutant protein of claim 2, wherein RSV subtype A is an RSV A2 strain or a long chain RSV strain. 4. The RSV F mutant protein of claim 2, wherein RSV subtype B is strain RSV 18537. 5. Mutant RSV F protein according to any one of paragraphs. 1-4, wherein the mutant RSV F protein contains an amino acid sequence that is 85% or more identical to SEQ ID NO: 2, wherein a leucine corresponding to leucine at position 141 of amino acid sequence SEQ ID NO: 1, or a leucine corresponding to leucine at position 142 amino acid sequence SEQ ID NO: 1, and leucine corresponding to leucine at position 373 of amino acid sequence SEQ ID NO: 1, are replaced by cysteines to form a disulfide bond between the cysteines, and thereby have the ability to induce antibody against the RSV type F pre-protein. 6. Mutant RSV F protein according to any one of paragraphs. 1-4, wherein the amino acid forming the furin recognition site, which is present on the C-terminal side of the pep27 region, is replaced so that the furin recognition site is not recognized by furin. 7. Mutant protein F RSV according to claim 6, where: the amino acid forming the furin recognition site is replaced by a non-basic amino acid, and the amino acid forming the furin recognition site is an amino acid selected from the group consisting of arginine corresponding to arginine at position 133 of the amino acid sequence SEQ ID NO: 1, arginine corresponding to arginine at position 135 amino acid sequence SEQ ID NO: 1, and arginine corresponding to arginine at position 136 amino acid sequence SEQ ID NO: 1. 8. The mutant RSV F protein of claim 6 or 7, wherein the mutant RSV F protein comprises an amino acid sequence wherein, in an amino acid sequence that is 85% or more identical to SEQ ID NO: 3, wherein is a leucine corresponding to leucine at amino acid position 141 sequences of SEQ ID NO: 1, or a leucine corresponding to leucine at position 142 of amino acid sequence SEQ ID NO: 1, and a leucine corresponding to leucine at position 373 of amino acid sequence SEQ ID NO: 1, replaced by cysteines and, in addition, an amino acid selected from a group consisting of an arginine corresponding to arginine at position 133 of amino acid sequence SEQ ID NO: 1, an arginine corresponding to arginine at position 135 of amino acid sequence SEQ ID NO: 1, and an arginine corresponding to arginine at position 136 of amino acid sequence SEQ ID NO: 1, replaced by a non-basic amino acid, and a disulfide bond is formed between the cysteines, and due to this, it has the ability to induce antibody against the RSV type F pre-protein. 9. The RSV F mutant protein according to claim 7 or 8, wherein the non-essential amino acid is asparagine. 10. Mutant RSV F protein according to any one of paragraphs. 1-9, wherein the threonine corresponding to the threonine at position 189 of amino acid sequence SEQ ID NO: 1 and/or the serine corresponding to the serine at position 190 of amino acid sequence SEQ ID NO: 1 are replaced by hydrophobic amino acids. 11. The RSV F mutant protein of claim 10, wherein each hydrophobic amino acid is independently selected from the group consisting of valine, isoleucine and leucine. 12. Mutant RSV F protein according to any one of paragraphs. 1-11, wherein the lysine corresponding to lysine at position 42 of amino acid sequence SEQ ID NO: 1 is replaced by arginine, and/or the valine corresponding to valine at position 384 of amino acid sequence SEQ ID NO: 1 is replaced by threonine. 13. An RSV F mutant protein having a mutation, wherein the mutation is a replacement of glutamic acid corresponding to glutamic acid at position 60 of the amino acid sequence SEQ ID NO: 1 with a non-acidic amino acid. 14. The RSV F mutant protein according to claim 13, wherein the non-acidic amino acid is selected from the group consisting of methionine, phenylalanine, leucine, threonine and serine. 15. RSV F fusion protein containing: mutant RSV F protein according to any one of claims. 1-14; And multimerization domain fused to the C-terminus of the mutant RSV F protein, where the multimerization domain is a foldon domain. 16. The RSV F fusion protein of claim 15, wherein the multimerization domain is a foldon domain derived from bacteriophage T4 fibritin. 17. The RSV F fusion protein of claim 15 or claim 16, wherein the multimerization domain is a foldon domain having the amino acid sequence of SEQ ID NO: 21. 18. The RSV F fusion protein according to any one of claims. 15-17, containing the amino acid sequence of any of SEQ ID NO: 10-13. 19. Multimer of mutant RSV F protein containing: two or more fusion proteins according to any one of claims. 15-18 linked via a multimerization domain. 20. The multimer according to claim 19, where the multimer is a trimer. 21. Particulate immunogenic product containing: particles of the mutant RSV F protein according to any one of claims. 1-14, where: mutant RSV F protein contains a particle-forming domain, two or more mutant RSV F proteins aggregate through a particle-forming domain to form a particle, said particle-forming domain is at a position closer to epitope I than to epitope Φ on the steric structure of the mutant protein, and said particle-forming domain is any from a peptide that binds to framework particles, a hydrophobic peptide chain and a self-associating protein. 22. Particulate immunogenic product containing: multimer particles according to claim 19 or 20, wherein the multimer contains a particle-forming domain, two or more multimers are aggregated by the particle-forming domain to form a particle, and said particle-forming domain is at a position closer to the I epitope than to the Φ epitope on the steric structure of the multimer, and said particle-forming domain The particle domain is any of a scaffold particle-binding peptide, a hydrophobic peptide chain, and a self-associating protein. 23. The immunogenic particulate product of claim 21 or 22, wherein two or more mutant RSV F proteins or multimers to produce an immunogenic particulate product, wherein the particulate domain is linked to the C-terminus of the mutant RSV F proteins or multimers , aggregate through a particle-forming domain. 24. Immunogenic product in the form of particles according to any one of paragraphs. 21-23, wherein the multimer is a trimer consisting of the RSV F fusion protein of claim 16. 25. The particulate immunogenic product of claim 24, wherein the particulate domain is an Fc domain consisting of the amino acid sequence of SEQ ID NO: 30, and wherein the particulate domain aggregates through binding to the Z domain of protein A immobilized on the VLP , derived from a modified HBs antigen. 26. The particulate immunogenic product of claim 24, wherein the particulate domain is a peptide consisting of the amino acid sequence SEQ ID NO: 42. 27. Use of an RSV F fusion protein to obtain an immunogenic particulate product containing: a mutant RSV F protein according to any one of claims. 1-14 or the RSV F fusion protein according to any one of claims. 15-18, and a particle-forming domain fused to its C-terminus, wherein the particle-forming domain is any of a scaffold-binding peptide, a hydrophobic peptide chain, and a self-associating protein. 28. Use of an RSV F fusion protein to produce the particulate immunogenic product of claim 27, wherein the particulate domain is an Fc domain consisting of the amino acid sequence of SEQ ID NO: 30. 29. Use of an RSV F fusion protein to produce the particulate immunogenic product of claim 27, wherein the particulate domain is a peptide consisting of the amino acid sequence of SEQ ID NO: 42. 30. A polynucleotide encoding the mutant RSV F protein according to any one of claims. 1-14. 31. A polynucleotide encoding an RSV F fusion protein according to any one of claims. 15-18. 32. An expression cluster containing a polynucleotide according to claim 30. 33. Expression cluster containing the polynucleotide according to claim 31. 34. A host cell comprising the expression cluster of claim 32, wherein the host cell expresses mutant RSV F proteins encoded by a polynucleotide from the expression cluster. 35. A host cell comprising the expression cluster of claim 33, wherein the host cell expresses RSV F fusion proteins encoded by a polynucleotide from the expression cluster. 36. An immunogen containing: mutant RSV F protein according to any one of paragraphs. 1-14, the RSV F fusion protein according to any one of claims. 15-18, multimer according to any one of paragraphs. 19-20 or an immunogenic product in the form of particles according to any one of paragraphs. 21-26. 37. Pharmaceutical composition for the prevention or treatment of RS viral infection, containing the expression cluster according to claim 32 or 33 or the immunogen according to claim 36. 38. An RSV vaccine containing the expression cluster of claim 32 or 33 or the immunogen of claim 36. 39. The pharmaceutical composition according to claim 37, which is intended for administration to humans. 40. The RSV vaccine according to claim 38, which is intended for administration to humans.

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